Method of screening

ABSTRACT

A method of detecting the presence of a functionally inhibitory immunointeractive molecule in a biological sample. Preferably, the immunointeractive molecule is directed to a pathogen derived antigen and, more particularly, a parasite derived antigen and, even more particularly, a  Plasmodium  drived antigen. The method of the present invention facilitates detection of the presence of functionally inhibitory immunointeractive molecules, both in vitro and in vivo, and is useful for qualitatively and/or quantitatively assessing the immune status of individuals who have been previously infected with a parasite, predicting the immune status of individuals vaccinated with an antigen based vaccine, determining the relative contribution of a specific immunoreactivity of antibody to the total inhibitory antibody elicited by combination vaccines which include two or more antigens, assessing vaccines to determine the efficacy of different forms of an antigen, determining vaccine potency, assessing the protective potential of certain immunoreactivities of antibodies and determining the importance of parasite inhibitory antibodies.

FIELD OF THE INVENTION

The present invention relates generally to a method of detecting thepresence of an immunointeractive molecule in a biological sample. Moreparticularly, the present invention relates to a method of detecting thepresence of a functionally inhibitory immunointeractive molecule in abiological sample. Preferably, said immunointeractive molecule isdirected to a pathogen derived antigen and, even more particularly, aparasite derived antigen. The method of the present inventionfacilitates detection of the presence of functionally inhibitoryimmunointeractive molecules, both in vitro and in vivo, and is useful,inter alia, for qualitatively and/or quantitatively assessing the immunestatus of individuals who have been previously infected with a parasite,predicting the immune status of individuals vaccinated with an antigenbased vaccines, determining the relative contribution of a specificimmunoreactivity of antibody to the total inhibitory antibody elicitedby combination vaccines which include two or more antigens, assessingvaccines to determine the efficacy of different forms of an antigen,determining vaccine potency, assessing the protective potential ofcertain immunoreactivities of antibodies and determining the importanceof parasite inhibitory antibodies.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Infection by the protozoan parasite Plasmodium falciparum results inseveral hundred million clinical cases of malaria each year of whichapproximately two million are fatal. the development of a malariavaccine is now a major global initiative. Progress toward this goalrequires an understanding of the mechanisms that underpin both naturallyacquired and vaccine-induced immunity. Antibodies that inhibit thegrowth of bloodstage P. falciparum parasites in vitro are found in thesera of some, but not all, individuals living in malaria endemic regions(Marsh, K., Otoo, L., Hayes, R. J., Carson, D. C. and Greenwood, B. M.(1989) Trans. R. Soc. Trop. Med Hyg 83:293-303; Brown, G. V., Anders, R.F., Mitchell, G. F. and Heywood, P. F. (1982) Nature 297:591-593; Brown,G. V., Anders, R. F. and Knowles, G. (1983) Infect. Immun. 39:1228-1235;Bouharoun-Tayoun, H., Attanath, P., Sabchareon, A., Chongsuphajaisiddhi,T. and Druilhe, P. (1990) J. Exp. Med. 172:1633-1641). Inhibitoryantibodies are likely to contribute to the clinical immunity observed inhighly exposed individuals but their overall significance to protectionremains unclear (Mohan, K. and Stevenson, M. M. (1998) Acquired immunityto asexual blood stages. In Malaria, parasite Biology, Pathogenesis andProtection. I. W. Sherman, editor. ASM Press, Washington, D.C. 467-493;McGregor, I. A. and Wilson, R. M. J. (1988) Specific immunity acquiredin man. In Malaria, Principles and Practices of Malariology. W. H.Wernsdorfer and I. A. McGregor, editors. Churchill Livingston, Inc., NewYork. 559-619).

Inhibitory antibodies function by preventing invasion of red blood cellsby the extracellular merozoite form of the parasite. A number ofmerozoite antigens have been shown to be targets of invasion inhibitoryantibodies including some that localize to the merozoite surface,parasitophorous vacuole, and apical organelles. One target of inhibitoryantibodies is the membrane-associated 19-kD COOH-terminal fragment ofmerozoite surface protein (MSP)′-1₁₉, a molecule that is now a leadingmalaria vaccine candidate (Digs, C. L., Ballou, W. R. and Miller, L. H.(1993) Parasitol. Today. 9:300-302; Good, M. F., Kaslow, D. C. andMiller, L. H. (1998) Annu. Rev. Immunol. 16:57-87). MSP-1₁₉ is unknown,however, allelic replacement experiments have shown that the function ofmost of the two EGF domains is conserved across distantly relatedPlasmodium species (O'Donnell, R. A., Saul, A., Cowman, A. F. and Crabb,B. S. (2000) Nat. Med. 6:91-95). The MSP-1₁₉ EGF domains formreduction-sensitive epitopes that are recognised by invasion-inhibitorymonoclonal and polyclonal antibodies (O'Donnell, R. A. et al. 2000supra; Blackman, M. J., Heidrich, H.-G., Donachie, S., McBridge, J. S.and Holder, A. A. (1990) J. Exp. Med 172:379-382; Chappel, J. A. andHolder, A. A. (1993) Mol. Biochem. Parasitol. 60:303-311; Cooper, J. A.,Cooper, L. T. and Saul, A. J. (1992) Mol. Biochem. Parasitol.51:301-312; Chang, S. P., Gibson, H. L., Lee Ng, C. T., Bar, P. J. andHui, G. S. (1992) J. Immunol. 149:548-555). MSP-1₁₉-specific inhibitoryantibodies are also present in the sera of individuals naturally exposedto P. falciparum (Egan, A., Burghaus, P., Druilhe, P., Holder, A. andRiley, E. (1999) Parasite Immunol. 21:133-139). These antibodiesrecognise epitopes formed by the double EGF domain and by the second EGFdomain alone (Egan, A., Burghaus, P., Druilhe, P., Holder, A. and Riley,E. (1999) Parasite Immunol. 21:133-139). The mechanism of inhibition byMSP-1₁₉ antibodies is not fully understood, however, those that preventthe secondary processing of a precursor molecule and hence the formationof MSP-1₁₉ also effectively inhibit merozoite invasion of RBCs(Blackman, M. J., Scott Finnigan, T. J., Shai, S. and Holder, A. A.(1994) J. Exp. Med. 180:389-393).

In light of the extensive research and development which is now directedto the development of a malaria vaccine, it is clearly necessary thatthere are available rapid and accurate methods of qualitatively and/orquantitatively screening for the presence of an immune response to thisparasite. To date, most such screening assays have been based on ananalysis of the presence of antibody molecules based on binding of theantibody to the pathogen of interest. These results have been obtainedusing methods such as ELISA. However, such assays do not discriminatebetween the presence of immunointeractive molecules which bind but whichdo not further impact on the functional activity of the pathogen towhich they bind versus immunointeractive molecules which do impact onthis functioning. For example, it is known that some specificities ofantibodies which are generated to the malaria MSP-1₁₉ antigen do notimpact on the functional activity of the parasite, while others do. Suchfunctionally inhibitory antibodies are particularly useful because inaddition to facilitating the induction of various antibody relatedclearance mechanisms, they down-regulate the functional activity of themalaria parasite by, inter alia, inhibiting its ability to infect redblood cells. Clearly, where one is seeking to induce an immune responsewhich either inhibits or clears a malaria infection, it is desirable tofocus on the induction of an immune response which is inhibitory to theviability, functioning and/or proliferation of the parasite in additionto facilitating the induction of traditional clearance mechanisms.Further, to the extent that the generation of such an immune response isessential in order to achieve effective immunity, it is necessary thatone has access to screening assays which can detect and measure thequality of an immune response which an individual has generated.

Accordingly, there is a need to develop more sophisticated screeningassays which are able to analyse the immunointeractive moleculecomponent of a biological sample at both the qualitative andquantitative levels, in particular, assays which are able to analyse thefunctional impact of an immunointeractive molecule on a given pathogen.

In work leading up to the present invention, the inventors havedeveloped a means of detecting the presence of a functionally inhibitoryimmunointeractive molecule, in particular an antibody, as opposed tomerely detecting the absolute levels of an immunointeractive molecule onthe basis of binding specificity alone. This objective is achieved byanalysing a functional pathogen parameter, such as pathogen growth forexample, of a pathogen expressing the native form of the antigen ofinterest, which pathogen has been contacted with the purportedimmunointeractive molecule sample, relative to that of a pathogen whichhas been genetically altered such that it expresses an epitopicallydifferent form of the antigen in issue. Where inhibition of the subjectfunctional parameter is observed in cultures of the pathogen expressingthe native form of antigen relative to the genetically altered cultures,there is indicated the presence of functionally inhibitory antibody inthe sample. However, where no difference is observed in the functionaloutput of the native antigen cultures relative to the geneticallyaltered cultures, there is indicated the absence of functionallyinhibitory antibody (despite the fact that standard immunointeractivitybased assays such as ELISAS may indicate the presence ofimmunointeractive molecules, such as antibodies, which are neverthelessbinding to the pathogen in issue). The development of thiscorrelate-of-protection assay now facilitates the analysis of thequality of an immune response. Further, these developments havefacilitated the development of both in vitro and in vivo based screeningmethods.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

One aspect of the present invention provides a method of detecting thepresence of a functionally modulatory immunointeractive molecule in abiological sample, which immunointeractive molecule is directed to apathogen derived antigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein modulation in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory immunointeractive molecule in    said sample.

Another aspect of the present invention provides a method of detectingthe presence of a functionally inhibitory immunointeractive molecule ina biological sample, which immunointeractive molecule is directed to apathogen derived antigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein a decrease in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory immunointeractive molecule in    said sample.

Yet another aspect of the present invention provides a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to a pathogen derivedantigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein a decrease in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory antibody in said sample.

Still another aspect of the present invention provides a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to a parasite derivedantigen, said method comprising:

-   (i) contacting a parasite expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a parasite expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the parasites of    step (i) and step (ii)    wherein a decrease in the functional activity of the parasite of    step (ii) relative to the parasite of step (i) is indicative of the    presence of a functionally inhibitory antibody in said sample.

In still yet another aspect there is provided a method of detecting thepresence of a functionally inhibitory antibody in a biological sample,which antibody is directed to a Plasmodium derived antigen, said methodcomprising:

-   (i) contacting Plasmodium expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immuno interaction;-   (ii) contacting Plasmodium expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) assessing the level of inhibition of red blood cell    invasiveness of Plasmodium of step (i) and step (ii)    wherein a decrease in the red blood cell invasiveness of the    Plasmodium of step (ii) relative to the Plasmodium of step (i) is    indicative of the presence of a functionally inhibitory antibody in    said sample.

In yet still another aspect there is provided a method of detecting thepresence of a functionally inhibitory antibody in a biological sample,which antibody is directed to Plasmodium falciparum MSP-1, said methodcomprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of MSP-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of MSP-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii) wherein a decrease in the functional activity    of the Plasmodium falciparum of step (ii) relative to the Plasmodium    falciparum of step (i) is indicative of the presence of a    functionally inhibitory antibody in said sample.

Preferably, said MSP-1 is the block 17 C-terminal domain or the block 2N-terminal domain of MSP-1.

In a further aspect there is provided a method of detecting the presenceof a functionally inhibitory antibody in a biological sample, whichantibody is directed to Plasmodium falciparum AMA-1, said methodcomprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of AMA-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of AMA-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said AMA-1 is domain 3 of AMA-1.

In yet another further aspect there is provided a method of detectingthe presence of a functionally inhibitory antibody in a biologicalsample, which antibody is directed to any one or more of Plasmodiumfalciparum MSP-2, MSP-3, MSP-4 and/or MSP-5, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of MSP-2, MSP-3, MSP-4 and/or MSP-5 with said sample    for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of MSP-2, MSP-3, MSP-4 and/or MSP-5 with said sample for    a time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

In still yet another further aspect there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumRAP-2 and/or RAP-1, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of RAP-2 and/or RAP-1 with said sample for a time and    under conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of RAP-2 and/or RAP-1 with said sample for a time and    under conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii) wherein a decrease in the functional activity    of the Plasmodium falciparum of step (ii) relative to the Plasmodium    falciparum of step (i) is indicative of the presence of a    functionally inhibitory antibody in said sample.

Preferably, said RAP-1 is the N-terminal region of RAP-1.

In a further aspect there is provided a method of detecting the presenceof a functionally inhibitory antibody in a biological sample, whichantibody is directed to Plasmodium falciparum erythrocyte bindingantigen, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of erythrocyte binding antigen with said sample for a    time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of erythrocyte binding antigen with said sample for a    time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said erythrocyte binding antigen is EBA-175 and even morepreferably the F2 domain of EBA-175.

In another further aspect there is provided a method of detecting thepresence of a functionally inhibitory antibody in a biological sample,which antibody is directed to Plasmodium falciparum CSP, said methodcomprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of CSP with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of CSP with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said CSP-1 is the block 2 N-terminal domain of CSP-1.

Another aspect of the present invention provides a method of detectingthe presence of a functionally inhibitory antibody in a biologicalsample, which antibody is directed to Plasmodium falciparum MSP-1₁₉,said method comprising:

-   (i) contacting a Plasmodium falciparum schizont of strain D10-PcM3′    with said sample for a time and under conditions sufficient to    facilitate immunointeraction;-   (ii) contacting a Plasmodium falciparum schizont of the strain D10    with said sample for a time and under conditions sufficient to    facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    falciparum strains of step (i) and step (ii);    wherein a decrease in the functional activity of the Plasmodium    falciparum strain of step (ii) relative to the Plasmodium falciparum    strain of step (i) is indicative of the presence of a functionally    inhibitory antibody in said sample.

In another aspect there is provided a method of detecting the presenceof a functionally inhibitory antibody in a biological sample, whichantibody is directed to Plasmodium falciparum MSP-1₁₉, said methodcomprising:

-   (i) contacting a Plasmodium falciparum schizont of strain D10 with    said sample for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting a Plasmodium falciparum schizont of strain    D10-PcMEGF with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    falciparum strains of step (i) and step (ii);    wherein a decrease in the functional activity of the Plasmodium    falciparum strain of step (ii) relative to the Plasmodium falciparum    strain of step (i) is indicative of the presence of a functionally    inhibitory antibody in said sample.

Accordingly, in yet another embodiment there is provided a method ofdetecting the presence of a functionally inhibitory antibody in apopulation of mice, which antibody is directed to Plasmodium falciparumMSP-1 and which method is performed in vivo in said mice, said methodcomprising:

-   (i) introducing to at least one of said mice a wild-type Plasmodium    berghei for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) introducing to at least one of said mice, other than the mouse    of step (i), a Plasmodium berghei strain, which strain expresses the    Plasmodium falciparum MSP-1 block 17C-terminal domain, for a time    and under conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    berghei of step (ii) relative to the Plasmodium berghei of step (i)    is indicative of the presence of said functionally inhibitory    antibody in said mice.

Most preferably, there is provided a method of detecting the presence ofa functionally inhibitory antibody in a population of mice, whichantibody is directed to Plasmodium falciparum MSP-1₁₉ and which methodis performed in vivo in said mice, said method comprising:

-   (i) introducing to at least one of said mice a wild-type Plasmodium    berghei for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) introducing to at least one of said mice, other than the mouse    of step (i), a Plasmodium berghei schizont of the strain Pb-PfM19    for a time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    berghei strains of step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    berghei strain of step (ii) relative to the Plasmodium berghei    strain of step (i) is indicative of the presence of said    functionally inhibitory antibody in said mouse.

In still another aspect the present invention is directed to a method ofassessing the nature of an immune response to an antigen in accordancewith the methods defined hereinbefore.

In yet another aspect, the present invention extends to the pathogensdefined herein.

Accordingly, yet another aspect of the present invention is directed toan isolated pathogen, which pathogen expresses a non-wild-type form ofone or more antigens derived from said pathogen.

More particularly, the present invention provides an isolated malariapathogen, which pathogen expresses a non-wild-type form of one or moreantigens derived from said pathogen.

Preferably, the present invention provides an isolated Plasmodium, whichPlasmodium expresses a non-wild-type form of MSP-1.

More preferably, said MSP-1 is the block 17 C-terminal domain or theblock 2 N-terminal domain of MSP-1.

Most preferably, said Plasmodium is Plasmodium berghei expressing thePlasmodium falciparum form of the MSP-1₁₉ antigen.

Even more preferably, said Plasmodium berghei is the Pb-PfM19 strain.

In another embodiment, the present invention provides an isolatedPlasmodium pathogen expressing a non-wild-type form of one or moreantigens derived from said pathogen, which antigens are selected fromthe list of:

-   (i) the apical membrane domain (AMA-1)-   (ii) merozoite surface protein 2, 3, 4 and/or 5 (MSP-2, MSP-3, MSP-4    and/or MSP-5)-   (iii) rhoptry associated protein 2 (RAP-2)-   (iv) erythrocyte binding antigens (EBA-175)-   (v) circumsprozoite antigen (CSP)

In yet another aspect, the present invention extends to the pathogensdefined herein when used in accordance with the method of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is An image showing generation of a transfected P. falciparumline containing the complete MSP-1₁₉ EGF domains from P. chabaudi inplace of the endogenous molecule. (A) Alignment of MSP-1₁₉ sequencesfrom P. falciparum (MAD20 allele; GenBank/EMBL/DDBJ accession no.M19143) and P. chabaudi (adami DS line; GenBank/EMBL/DDBJ accession no.AF149303). The arrows indicate the sites of secondary cleavage,asterisks denote identical residues, and dots highlight conservedresidues. The disulfide bonds expected for EGF-like domains are shown(black lines). Note the absent disulfide bond in P. chabaudi (dashedline). The nature of the gene fusions in the various MSP-1 hybrid linesis represented underneath the alignment with the dashed linerepresenting endogenous P. falciparum sequence and the solid line, P.chabaudi sequence. (B) The plasmid pPcMEGF was constructed by ligating aDNA fragment containing P. falciparum MSP-1 sequence (Target) fused tosequence encoding MSP-1₁₉ from P. chabaudi MSP-1 (dark shading) into theXhoI site of pHC2. The predicted structure of the MSP-1 loci followingintegration of pPcMEGF and the location of the XbaI (X) sites used tomap these loci are shown. The location of XbaI sites unique to D10-PcM3′and D10-PfM3′ are bracketed and are represented as X.Pc and X.Pf,respectively. All sizes are to scale with the exception of the plasmidbackbone (dashed line). (C) Southern blot analysis of gDNA restrictedwith XbaI showing that pPcMEGF had integrated into MSP-1 as predictedand that the resultant line (D10-PcMEGF) differs from the previouslyestablished lines D10-PfM3′ and D10-PcM3′ (O'Donnell, R. A. et al.,2000). The 0.9-kb PfMSP-1 fragment (Target) was used to probe the blot.

FIG. 2 is an image of transfected D10-PcMEGF parasites express afunctional MSP-1 chimera. (A) Western blot analysis of parasite proteinsfrom extracted enriched schizont (Schiz) or merozoite (Mer) preparationsof parental D10 and the D10-PcMEGF clones (PcMEGF.1 and PcMEGF.2).Proteins were separated by SDS-PAGE under non-reducing conditions,transferred to PVDF membranes, and probed with either 4H9/19 or *PcM19antibodies as indicated. The position of molecular weight standards areshown to the left and are in Kd. (B) Localization of MSP-1 expressed inthe transfected lines by indirect IFA. D10-PfM3′ (PfM3′) and D10-PcMGF.1(PcMEGF.1) schizont-stage parasites were incubated with a mixture of4H9/19 and *PcM19 antibodies. After incubation in the presence of amixture of FITC-conjugated anti-mouse and rhodamine-conjugatedanti-rabbit Igs, parasites were visualised by microscopy. Originalmagnification: 1,000×. the same fields were photographed underfluorescence conditions to detect the FITC or rhodamine fluorochromes.(C) In vitro inhibition assays of D10, D10-PcM3′, and D10-PcMEGF clones(PcMEGF.1 and PcMEGF.2) with different concentrations of *PcM19antibodies (IgG). Error bars represent SDs.

FIG. 3 is a graphical representation of the invasion inhibition oftransfected P. falciparum parasites expressing divergent MSP-1₁₉ domainsby sera from clinically immune individuals reveals an important role forMSP-1₁₉-specific antibodies. (A) Assay 1, microscopy. Microscopy-basedinvasion inhibition assay involving the detection of ring-stage D10 andD10-PcMEGF (PCMEGF) parasites after cultivation in the presence of eachindividual serum. (B) Assay 2, hypoxanthine uptake. Alternative,invasion-inhibition assay comparing D10-PfM3′ (PfM3′) and D10-PcMEGFparasites using [³H]hypoxanthine uptake as a measure of parasite growth.Invasion is represented as either parasiternia (A) or counts (B) and isexpressed as a percentage of the invasion observed in parasites culturedin negative control sera (HNIS). The means of samples within a serum setagainst each parasite line are indicated. P values from a Student's ttest comparing the means in each panel are shown.

FIG. 4 is a graphical representation of the invasion-inhibition assaywith representative individual sera from PNG-B and Pc-immune serum setsagainst D10-PfM3′ and D10-PcMEGF parasite lines. Samples were selectedfrom assay 2 and represent typical examples of the inhibitory activitiesobserved. The results obtained for the control sera in assay 2, anti-P.falciparum AMA-1 (*PfAMA1), and *PcM19 IgG are shown. Error barsrepresent the range observed in duplicate samples.

FIG. 5 is an image of co-cultivation of D10-PfM3′ and D10-PcMEGFparasites in the presence of immune sera confirms an important role forMSP-1₁₉ antibodies in invasion inhibition. Ring-stage D10-PfM3′ andD10-PcMEGF parasites were combined at an equal ratio and cultured in thepresence of the pooled sera indicated at right. Smears from days 1 and 5were analyzed by double-labelling IFA. Mature stage (pigmented) greenand red parasites were counted in 16 fields each containing at least 10parasites. The same fields were observed by fluorescence microscopyusing filters to detect the FITC or rhodamine fluorochromes. Results areexpressed as a ratio of D10-PfM3′ to D10-PcMEGF. A representative fieldof parasites (at day 3) cultured in the presence of HNIS pool is shown(inset).

FIG. 6 is an image of the functional complementation of divergentMSP-1₁₉ domains in vivo: Replacement of the P. berghei MSP-119 domainwith that from P. falciparum MSP-1₁₉ (MAD20 allele) in P. bergheiparasites cultured in mice. (A) Schematic diagram showing the P. bergheiMSP-1 locus before (top) and after (bottom) homologous integration ofthe pPb-PfM19 transfection plasmid. Within the plasmid, the location ofthe 5′ and 3′ homologous sequences used for gene targeting (solidlines), the P. falciparum MSP-1₁₉ sequence (black box), the HSP863′region (3′) and the selectable marker (Tg DHFR-TS cassette) are shown.The location of HincII (H), EcoRI (E) and SwaI (S) restriction sites areshown. (B) Southern blot of HincII (left) or EcoRI/SwaI (right) digestedgenomic DNA from wild type P. berghei (Pb WT) or transfected P. berghei(Pb-PfM19). The location of the probe is shown (solid dashed line). Thepresence of bands of the expected sizes and the absence of an endogenouswil type band in the Pb-PfM19 lanes is indicative of a pure populationof transfected possessing the expected double-crossover homologousintegration event.

This data demonstrates that rodent malaria parasites (P. berghei) areviable in mice when expressing a MSP-1 hybrid molecule incorporating thecomplete MSP-1₁₉ domain from P. falciparum in place of endogenous P.berghei MSP-1. This is the first demonstration that these domains arefunctionally conserved across divergent Plasmodium species in in vivocultured blood-stage parasites.

FIG. 7 is a schematic representation of P. berghei and P. falciparumMSP-1 chimeras. The MSP-1 sequences of P. berghei (grey), P. falciparum(red) and P. chabaudi (blue) are represented. The Pb-PbM19 controlchimera (this study) is identical at the MSP-1 locus to wildtype P.berghei, whereas the Pb-PfM19 chimera (this study) expresses P.falciparum MSP-1₁₉ in place of the endogenous molecule. Likewise,D10-PfM3′ (21), is identical at the MSP-1 locus to wildtype P.falciparum, while D10-PcMEGF expresses the P. chabaudi MSP-1₁₉polypeptide (9). The arrows indicate the MSP-1 secondary cleavage site.

FIG. 8 is a schematic representation of the generation of P. bergheichimera lines containing either P. berghei or P. falciparum MSP-1₁₉. (A)Schematic diagram of the P. berghei MSP-1 locus, the transfection vector(pPb-PfM19) used to replace the endogenous MSP-1₁₉ molecule, and thepredicted MSP-1 locus of the Pb-PfM19 chimeric line after integration.The grey box represents endogenous P. berghei MSP-1₉ sequence while theblack box represents P. falciparum MSP-1₁₉ sequence. The solid lines inpPb-PfM19 depict targeting sequence used to drive integration. The samestrategy was used to create the Pb-PbM19 chimeric line, with theexception that the sequence represented by the black box is that of P.berghei MSP-1₁₉. Tg DHFR-TS, selectable marker cassette; 3′, HSP86 3′UTR. The expected sizes of fragments resulting from digestion witheither HincII (H) or PstI (P) are shown. (B) Southern blot analysis ofdigested genomic DNA from P. berghei wiltype and chimeric lines.Replacement of the endogenous MSP-1₁₉ sequence with that of P.falciparum (Pb-PfM19 chimera) or wiltype P. berghei sequence (Pb-PbM19chimera) was confirmed by the hybridisation of Southern blots witheither probe A or B as shown in FIG. 2A.

FIG. 9 is an image of the phenotypic analysis of P. berghei chimericlines. (A) Western blot analysis of late stage parasite extracts usingrabbit αPbM19 or αPfM19 antibodies (both diluted 1/4000) demonstratesthat both full-length MSP-1 (approximately 200 kDa) and MSP-1₁₉(approximately 19 kDa) could be detected in wildtype and chimeric P.berghei lines. (B) Localisation of MSP-1₁₉ in wildtype and chimeric P.berghei lines by indirect immunofluorescence assay. Schizont-stageparasites were incubated with a mixture of αPbM19 ( 1/1000) and 4H9/19 (1/100) antibodies, followed by a mixture of FITC-conjugated anti-rabbitand rhodamine-conjugated anti-mouse immunoglobulins (both diluted1/200). The same fields were photographed under fluorescence conditionsto detect the FITC or rhodamine fluorochromes. (C) Course of bloodparasitemia in mice following infection at Day 0 with P. bergheiwildtype, Pb-PbM19 or Pb-PfM19. Shown is the mean±SD of the parasitemiaobserved in 5 mice.

FIG. 10 is a graphical representation of mice repeatedly infected withP. berghei transfectants eliciting MSP-1₁₉ specific inhibitoryantibodies. (A) Anti-MSP-1₁₉ antibody endpoint titres of serum fromPb-PfM19 and Pb-PbM19 immune mice against recombinant P. falciparum andP. berghei MSP-1₁₉—GST fusion proteins. (B) Invasion inhibition assay ofD10-PfM3′ and D10-PcMEGF parasite lines in the presence of individualserum from Pb-PfM19 and Pb-PbM19 immune mice. The invasion rate isexpressed as a percentage of the invasion observed in parasites culturedin human non-immune sera (HNIS). The numbers shown represent the P.falciparum MSP-1₁₉ specific invasion inhibitory activity of a givenserum, calculated by subtracting the invasion rate of D10-PfM3′ fromthat of D10-PcMEGF.

FIG. 11 is a graphical representation of the evidence that MSP-1₁₉specific inhibitory antibodies control a blood-stage infection. MSP-1₁₉specific invasion inhibitory activity of serum from individual Pb-PfM19immune mice plotted against the log of the peak parasitemia attainedafter challenging corresponding mice with Pb-PfM19.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatfunctionally inhibitory immunointeractive molecules, such as antibodies,can be both qualitatively and quantitatively identified where onemeasures a functional parameter of the pathogen of interest, whichfunctional parameter is that which would be down-regulated and/orinhibited in the presence of the immunointeractive molecule of interest,and where one analyses this parameter relatively to that of agenetically altered pathogen which expresses an epitopically distinctform of the antigen which is the target of the immunointeractivemolecule of interest. This determination has now facilitated thedevelopment of in vitro and in vivo assays directed to screening forimmunointeractive molecules (in particular, antibodies) which, inaddition to binding to a pathogen, also act to inhibit or otherwisedown-regulate one or more of the pathogen's functional attributes.Although the method of the present invention is exemplified with respectto malaria, it can be applied to any pathogen and now facilitates theanalysis of the functional quality of an immune response, which type ofanalysis was not previously available. The method of the presentinvention is applicable in a range of situations including, but notlimited to, the assessment of the quality of an individual's immunity,the determination of whether a vaccine protocol is inducing afunctionally relevant form of immunity or to determining the relativecontribution of a specific immunointeractive molecule to the totalinhibitory functioning of a given immune response.

Accordingly, one aspect of the present invention provides a method ofdetecting the presence of a functionally modulatory immunointeractivemolecule in a biological sample, which immunointeractive molecule isdirected to a pathogen derived antigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein modulation in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory immunointeractive molecule in    said sample.

Reference to “modulation” should be understood as a reference toup-regulation or down-regulation. Although the preferred method is todetect immunointeractive molecules which down-regulate functionalactivity, there may be circumstances in which it is desirable ornecessary to screen for molecules which aberrantly, or otherwise, act toup-regulate the functional activity of a pathogen.

Accordingly, the present invention more particularly provides a methodof detecting the presence of a functionally inhibitory immunointeractivemolecule in a biological sample, which immunointeractive molecule isdirected to a pathogen derived antigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein a decrease in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory immunointeractive molecule in    said sample.

The inventors in respect of the present invention have actuallydeveloped two aspects in respect of this method of screening, thesebeing its application in an in vitro environment and its application inan in vivo environment. The development of an in vivo assay, inparticular, is a surprising and unusual development which nowfacilitates forms of analysis which were not previously available, andwhich would not be available even utilising the in vitro methodologyherein described.

In relation to the in vivo methodology which is disclosed herein, theinventors have specifically developed a non-human animal model methodfor detecting, in vivo, functionally inhibitory antibodies. This methodis based on the determination that the infection of a non-human animalwith a pathogen expressing an antigen of interest, which pathogen is onewhich is both suitable for colonising the selected animal model and theactivity of which will be modulated if bound by a functionallyinhibitory antibody directed to said antigen, provides a means fordetermining whether a biological sample which is introduced to saidanimal comprises functionally inhibitory molecules. This determinationis based on a relative analysis of the functionality of pathogensexpressing the native form of the antigen of interest versus thoseexpressing an epitopically distinct form of said antigen.

It should be understood that such an in vivo detection method hasextensive application. For example, in one embodiment, a murine model isinfected with a murine malaria parasite expressing a form of the epitopeof interest which is expressed by a human malaria parasite. There isthereby provided a means of screening a biological sample for thegeneration and/or presence of functionally inhibitory antibodies,directed to the human form of the epitope, which antibodies do not bindto the epitopically distinct murine homologue of the epitope. The personof skill in the art would recognise that the availability of such an invivo screening model provides advantages which are not provided by an invitro based screening assay, such as the ability to immunise the animalmodel with developmental vaccines which are designed to elicit an immuneresponse to the epitope of interest. In this way, vaccines intended foruse in humans can be trialed in a non-human model which will provideaccurate results in respect of the quality of the immune response whichis generated to that vaccine, in terms of its functional impact of theepitope of interest.

As would be appreciated by the person of skill in the art, thedevelopment of the in vitro assay herein described herein also providesunique advantages, such as the capacity to rapidly perform highthroughput analysis.

Reference to “immunointeractive molecule” should be understood as areference to any molecule which comprises an antigen binding portion. By“antigen” is meant any molecule against which an immune response may begenerated. In accordance with the method of the present invention, theantigen is a pathogen. It should be understood that the subjectimmunointeractive molecule may take any form. For example, it may be asecreted form of a molecule, such as an antibody, or it may be linked,bound or otherwise associated with any other molecule, such as a cell.For example, a T cell receptor is likely to be associated with a Thelper cell or a T cytotoxic cell. It should be understood that themolecule or cell may also be coupled to any other proteinaceous ornon-proteinaceous molecule, such as a tag which facilitates itsdetection or tracking. The immunointeractive molecule may be naturallyoccurring or it may have been genetically or otherwise modified.Examples of molecules contemplated by this aspect of the presentinvention include, but are not limited to, monoclonal and polyclonalantibodies (including synthetic antibodies), hybrid antibodies,humanised antibodies, catalytic antibodies and T cell antigen bindingmolecules. Preferably, said immunointeractive molecule is an antibody.Reference to “antibody” hereinafter is not intended to be limiting andshould be understood to include reference to any form ofimmunointeractive molecule.

The method of the present invention therefore still more particularlyprovides a method of detecting the presence of a functionally inhibitoryantibody in a biological sample, which antibody is directed to apathogen derived antigen, said method comprising:

-   (i) contacting a pathogen expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a pathogen expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the pathogens of    step (i) and step (ii)    wherein a decrease in the functional activity of the pathogen of    step (ii) relative to the pathogen of step (i) is indicative of the    presence of a functionally inhibitory antibody in said sample.

Reference to “pathogen” should be understood as a reference to anymicroorganism which can infect a human or non-human animal or to amolecule secreted therefrom. The subject pathogen may or may not resultin the onset of a disease condition. In this regard, many pathogens doinduce diseases. However, some pathogens can colonise an animal andexist in a symbiotic relationship without the onset of a diseasecondition. Such pathogens, due to their foreign nature, may neverthelessresult in the onset of an acute or chronic immune response, the analysisof which response in accordance with the methods defined herein may benevertheless desirable. Reference to “pathogen” should also beunderstood to encompass pathogens which have either naturally ornon-naturally undergone some form of mutation, genetic manipulation orany other form of manipulation. Accordingly, the chimaeric Plasmodiumfalciparum strains disclosed herein should be understood to fall withinthe scope of the definition of “pathogen”. Examples of pathogensinclude, but are not limited to, bacteria, viruses and parasites.Preferably, the subject pathogen is a parasite and even more preferablya malaria inducing parasite.

The human or non-human animal as described herein includes humans,primates, livestock animals (eg. sheep, pigs, cows, horses, donkeys),laboratory test animals (eg. mice, rats, rabbits, guinea pigs),companion animals (eg. dogs, cats), captive wild animals (eg. foxes,kangaroos, deer), aves (eg. chicken, geese, ducks, emus, ostriches),reptiles or fish. Preferably, the subject is a human.

The present invention therefore more preferably provides a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to a parasite derivedantigen, said method comprising:

-   (i) contacting a parasite expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting a parasite expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the parasites of    step (i) and step (ii)    wherein a decrease in the functional activity of the parasite of    step (ii) relative to the parasite of step (i) is indicative of the    presence of a functionally inhibitory antibody in said sample.

Preferably, the subject parasite is a malaria inducing parasite.

“Malaria” is a term used to describe a class of diseases which arecaused by infection with the protozoans of the genus Plasmodium. Thesediseases are also known by other names including Ague, Marsh Fever,Periodic Fever and Paludism. The Plasmodium species P. falciparum, P.malariae, P. ovale and P. vivax will each result in the onset of malariain the human. In general, and without limiting the present invention inany way, the disease is transmitted by the Anopheles mosquito and isconfined mainly to tropical and sub-tropical areas. Parasites in theblood of an infected person are taken up into the stomach of themosquito as it feeds. Here, they multiply and then invade the mosquitosalivary glands. When the mosquito bites a subject, parasites aresimultaneously injected into the blood stream and thereafter migrate tothe liver and other organs, where they multiply. After an incubationperiod varying from 12 days (P. falciparum) to 10 months (some varietiesof P. vivax), parasites return to the blood stream and invade the redblood cells. Rapid multiplication of the parasites results indestruction of the red cells and the release of more parasites capableof infecting other red cells. This causes a short bout of shivering,fever and sweating and the loss of healthy red cells results in anaemia.When the next batch of parasites is released, symptoms reappear. Theinterval between fever attacks varies in different forms of malaria. Forexample, in Quartan malaria the interval is approximately three days andis caused by the species P. malaria. In Tertian malaria, the interval istwo days and is caused by the species P. ovale and P. vivax. Inmalignant Tertian malaria, this being the most severe form of malaria,the interval is from a few hours to two days. This form of malaria iscaused by P. falciparum.

Still without limiting the present invention in any way, the primitivemalarial parasites which are injected by the mosquito are termedsporozoites. These sporozoites circulate in the blood for a short timeand then settle in the liver where they enter the parenchymal cells andmultiply. This stage is known as the pre-erythrocytic schizogony. Aftermultiplication, there may be thousands of young parasites known asmerozoites in one liver cell. At this time, the liver cell ruptures andthe free merozoites enter red blood cells. In the red blood cells, theparasites develop into two forms, a sexual and an asexual cycle. Thesexual cycle produces male and female gametocytes which circulate in theblood and are taken up by a female mosquito when taking a blood meal. Inthe asexual cycle, the developing parasites form schizonts in the redblood cells which contain many merozoites. The infected red cellsrupture and release a batch of young merozoites which invade new redcells. The species P. vivax, P. ovale and P. malariae develop in theperipheral blood subsequently to the liver cycles. However, in the caseof P. falciparum only ring forms and gametocytes are present in theperipheral blood.

Accordingly, it should be understood that many pathogens, in particular,parasites, pass through a number of developmental stages during theirlife cycle. Reference to “pathogen” in the context of the presentinvention and in particular in the context of steps (i) and (ii) asdefined herein, should therefore be understood as a reference to apathogen at any one of its life cycle developmental stage, whether thatbe a mature or immature developmental stage. For example, in the contextof the embodiment exemplified herein, being the screening for P.falciparum MSP-1₁₉-directed functionally inhibitory antibodies, the P.falciparum pathogen which is utilised in steps (i) and (ii) may be ofany suitable developmental stage. However, in accordance with thespecific exemplification provided herein, ring stage parasites aresynchronised and then allowed to mature through to thetrophozoite/schizont stages prior to culturing, in accordance with steps(i) and (ii), with the biological sample of interest. It should beunderstood, however, that although this is a preferred form ofconducting the subject screening test, the person of skill in the artmay seek to use parasites at any other developmental stage, depending onthe particular nature of the antigen against which immunointeractiveantibodies are to be detected.

The method of the present invention is directed to screening forfunctionally inhibitory immunointeractive molecules, in particularfunctionally inhibitory antibodies. By “functionally inhibitory” ismeant that the subject antibody, by virtue of binding, interacting orotherwise associating with a pathogen, acts to inhibit, prevent orotherwise down-regulate any one or more functional activities of thatpathogen such as, but not limited to, division, maturation or cellularinvasiveness. That is, the subject functional activity is inhibited byvirtue of the association of the pathogen with a functionally inhibitoryantibody, per se, and not necessarily by virtue of any subsequentclearance mechanism which may also occur (although such a possibility isnot excluded by the present invention). For example, and withoutlimiting the invention in any way, binding of certain antibodyspecificities to MSP-1₁₉ have been shown to prevent P. falciparummerozoites from further dividing and/or colonising red blood cells. Itis not fully understood how the antibody achieves this outcome.Nevertheless, such antibodies are clearly highly desirable in an immuneresponse since the traditional antibody based clearance mechanisms arenot always effective in clearing or even controlling, certain types ofparasitic infections.

In this regard, reference to assessing the level of “functionalactivity” of the pathogen should be understood as a reference toassessing the activity of the pathogen which corresponds to the activitywhich the functionally inhibitory antibody in issue would down-regulate.Although the present invention is exemplified in terms of screening forthe modulation of a single functional activity, it should be understoodthat the person of skill in the art may screen for any one or morefunctional activities, for example, either because the person of skillin the art is simultaneously screening for the presence of a combinationof functionally inhibitory antibodies or because the subjectfunctionally inhibitory antibodies down-regulate more than onefunctional activity of the target pathogen. In accordance with theexemplification provided herein, in one embodiment the functionallyinhibitory antibodies of interest is one which down-regulates the redblood cell invasiveness of Plasmodium falciparum merozoites.Accordingly, the functional activity which is the subject of screeningis the capacity of P. falciparum merozoites, which have been culturedtogether with a test serum source to invade red blood cells.Nevertheless, it should be understood that a functionally inhibitoryantibody, as defined herein, may be additionally involved intraditionally understood immune clearance mechanisms. However, it is itsactivity as an inhibitor of one or more pathogen functional activitieswhich forms the basis of the detection of these antibodies in accordancewith the method of the present invention. To the extent that thepathogen of interest is Plasmodium, the subject functional inhibition ispreferably inhibition of red blood cell invasiveness.

Accordingly, there is provided a method of detecting the presence of afunctionally inhibitory antibody in a biological sample, which antibodyis directed to a Plasmodium derived antigen, said method comprising:

-   (i) contacting Plasmodium expressing an epitopically distinct form    of said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium expressing an epitopically native form of    said antigen with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (ii) assessing the level of inhibition of red blood cell    invasiveness of Plasmodium of step (i) and step (ii)    wherein a decrease in the red blood cell invasiveness of the    Plasmodium of step (ii) relative to the Plasmodium of step (i) is    indicative of the presence of a functionally inhibitory antibody in    said sample.

Preferably, said Plasmodium is Plasmodium falciparum.

As detailed hereinbefore, the method of the present invention detectsthe presence of a functionally inhibitory antibody based on an analysisof the functional activity of a pathogen which has been contacted with abiological sample of interest. In this regard, reference to “pathogenderived antigen” should be understood as a reference to the antigen towhich the subject functionally inhibitory antibody is directed. Itshould be understood that this antigen may form part of the pathogenitself or it may be a molecule which is secreted from the pathogen, theinteraction of which with a functionally inhibitory antibody, forexample, nevertheless acts to down-regulate one or more aspects of thefunctional activity of the pathogen itself or of that particularmolecule. The subject antigen may be one which is either permanently ortransiently expressed by the subject pathogen. The notion of transientexpression of an antigen is likely to be of particular relevance with apathogen such as a virus or parasite which passes through a number ofdistinct developmental life cycle stages.

It should be further understood that the subject antigen may compriseone or more epitopes, any one or more of which epitopes may berecognised by the antibody of interest. Alternatively, the subjectantigen may be a very small antigen and may, in its entirety, correspondto a single epitope. In general, any given antibody of interest wouldonly recognise one epitope of the antigen in issue, althoughcross-reactivity is nevertheless contemplated by the method of thepresent invention. The notion of an antibody expressing reactivitytowards a single epitope accords with accepted immunological principlesin relation to the specificity of antibody responses. In this regard,reference to the functionally inhibitory antibody being “directed” tothe antigen should be understood to mean that the antibody recognises anepitope which is present on the antigen. To the extent that saidpathogen is Plasmodium falciparum, said antigen is preferably any domainof MSP-1 (for example the “block 2” N-terminal domain or the block 17C-terminal domain), the apical membrane domain (AMA-1), merozoitesurface protein 2, 3, 4 and 5 (MSP-2, MSP-3, MSP-4 and MSP-5), rhoptryassociated protein 2 (RAP-2), RAP-1, erythrocyte binding antigens(EBA-175) or the circumsprozoite antigen (CSP).

Accordingly, in one preferred embodiment there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumMSP-1, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of MSP-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of MSP-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said MSP-1 is the block 17 C-terminal domain or the block 2N-terminal domain of MSP-1.

In another preferred embodiment there is provided a method of detectingthe presence of a functionally inhibitory antibody in a biologicalsample, which antibody is directed to Plasmodium falciparum AMA-1, saidmethod comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of AMA-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of AMA-1 with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said AMA-1 is domain 3 of AMA-1.

In yet another preferred embodiment, there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to any one or more ofPlasmodium falciparum MSP-2, MSP-3, MSP-4 and/or MSP-5, said methodcomprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of MSP-2, MSP-3, MSP-4 and/or MSP-5 with said sample    for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of MSP-2, MSP-3, MSP-4 and/or MSP-5 with said sample for    a time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

In still yet another preferred embodiment, there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumRAP-2 and/or RAP-1, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of RAP-2 and/or RAP-1 with said sample for a time and    under conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of RAP-2 and/or RAP-1 with said sample for a time and    under conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said RAP-1 is the N-terminal region of RAP-1.

In a further preferred embodiment, there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumerythrocyte binding antigen, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of erythrocyte binding antigen with said sample for a    time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of erythrocyte binding antigen with said sample for a    time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

Preferably, said erythrocyte binding antigen is EBA-175 and even morepreferably the F2 domain of EBA-175.

In another further preferred embodiment, there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumCSP, said method comprising:

-   (i) contacting Plasmodium falciparum expressing an epitopically    distinct form of CSP with said sample for a time and under    conditions sufficient to facilitate immunointeraction;-   (ii) contacting Plasmodium falciparum expressing an epitopically    native form of CSP with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    falciparum of step (ii) relative to the Plasmodium falciparum of    step (i) is indicative of the presence of a functionally inhibitory    antibody in said sample.

The method of the present invention overcomes previous shortcomings ofantibody based screening methods wherein only the presence or absence ofan antibody expressing a particular antigenic immunoreactivity could bemeasured. These methods (for example ELISAs, FACS analysis orimmunofluorescent microscopy) cannot distinguish between thoseantibodies of a particular immunoreactivity which can modulate thefunctional activity of the pathogen expressing that antigen versus thosewhich cannot. Even when analysing antibodies of differentimmunoreactivities, such methods cannot identify which of theseantibodies may additionally modulate pathogen functioning. The analysisof modulation of pathogen functioning is of particular importance wherethe antibody based clearance mechanisms which are up-regulated in aninfected individual upon the induction of a B cell response are not theonly therapeutic or prophylactic mechanism to provide a defence to thepathogen in issue. In particular, in relation to some diseaseconditions, the generation of antibodies which can interfere with thefunctioning of a pathogen provides significant protection above andbeyond that normally provided by antibody based clearance mechanisms,alone.

In order to enable the detection, monitoring and identification offunctionally inhibitory immunointeractive molecules, in particularantibodies, the inventors have designed an assay which screens both forspecific immunoreactivity and modulation of pathogen functioning. Thisis achieved by conducting a relative analysis of the level of functionalactivity of a pathogen, subsequently to its culture with the biologicalsample of interest, expressing the native form of the antigen ofinterest versus that of a pathogen expressing a form of the antigenwhich would not be recognised by the antibody in issue. Specifically,where there is observed a lower level of functional activity in nativepathogen cultures versus that observed in the epitopically distinctpathogen cultures, there is indicated the presence of antibody which isboth immunoreactive with the antigen which was rendered epitopicallydistinct in the control cultures and inhibitory of one or more of thefunctional activities of the pathogen of interest. Where the results donot demonstrate a difference in the level of functional activityexpressed by the pathogen in the two forms of cultures which areestablished, the results indicate that there is not present in thebiological sample any antibody which is directed to the antigen ofinterest and which modulates the functioning of the pathogen. Theseresults do not indicate, however, that there is not present antibody inthe culture which does recognise that epitope but which antibody doesnot modulate the functioning of the pathogen.

Reference to a biological sample “contacting” a pathogen of interestshould be understood as a reference to any method of facilitating theinteraction of any one or more components of the biological sample withthe pathogen, or molecules shed or secreted therefrom, such thatcoupling, binding or other association may occur. In this regard, itshould be understood that the method of the present invention may beperformed in vitro or in vivo. With respect to the in vitro applicationof this method, the biological sample and the pathogen of interest arepaced in contact in an artificial medium, such as a culture dish orflask. However, to the extent that the method of the present inventionis applied in vivo, the biological sample and the pathogen of interestwill be placed in contact within a biological organism such as ananimal. In this regard, it should be understood that the pathogen andthe biological sample may be separately or simultaneously introduced tothe animal model such that they contact one another within the animal.Alternatively, the pathogen and the biological sample may be placed intoinitial contact prior to their introduction to the host animal, forexample such that only one administration need be made to the animal.This form of administration should also be understood to fall within thescope of “contacting” as defined herein. Means of achieving such contactwould be well know to those of skill in the art.

Reference to an “epitopically native” form of the antigen should beunderstood to mean that the epitope which is recognised by the antibodyof interest is expressed by the pathogen either in its native/wild-typeform or in a form which comprises amino acid or other structural ornon-structural differences which do not impact on the ability of theantibody to recognise and bind the epitope. Reference to an“epitopically distinct” form of the subject antigen should be understoodto mean that the epitope which is recognised by the antibody of interesthas been altered such that it is no longer recognised and bound by theantibody of interest. The subject alteration can be achieved by any oneor more of a number of techniques which would be known to the person ofskill in the art including, but not limited to:

-   (i) deletion of the epitope from the antigen;-   (ii) replacement of the epitope with a homologous form of the    epitope which is not recognised by the antibody of interest;-   (iii) any one or more amino acid deletions, additions or    substitutions to the subject epitope;

In accordance with the exemplification provided herein, which isdirected to the identification of functionally inhibitory antibodiesdirected to the Plasmodium falciparum MSP-1₁₉ antigen, the MSP-1₁₉antigen of the Plasmodium falciparum merozoite is replaced with ahomologous form of the antigen which is not recognised by the antibodiesof interest. Specifically, the epitopically distinct form of Plasmodiumfalciparum is a genetically engineered form which expresses the MSP-1₁₉region from P. chabaudi, being the form of Plasmodium which infectsmice. In this regard, the strain of Plasmodium falciparum whichexpresses the native form of MSP-1₁₉ and which is exemplified herein isthe D10 strain. The P. falciparum parasites expressing divergent MSP-1₁₉domains are transfected D10 strain parasites which express the P.chabaudi domain (D10-PcMEGF). Whereas DIO-PcMEGF is a form of Plasmodiumfalciparum in which the entire EGF domains from MSP-1₁₉ are replacedwith those from P. chabaudi, the D10-PcM3′ strain of Plasmodiumfalciparum is one in which the Plasmodium parasite expresses a chimericform of MSP-1₁₉ in which approximately three quarters of the twoEGF-like domains that comprise MSP-1₁₉ are replaced with the equivalentdomains from the divergent rodent malaria P. chabaudi.

Accordingly, in a preferred embodiment there is provided a method ofdetecting the presence of a functionally inhibitory antibody in abiological sample, which antibody is directed to Plasmodium falciparumMSP-1₁₉, said method comprising:

-   (i) contacting a Plasmodium falciparum schizont of strain D10-PcM3′    with said sample for a time and under conditions sufficient to    facilitate immunointeraction;-   (ii) contacting a Plasmodium falciparum schizont of the strain D10    with said sample for a time and under conditions sufficient to    facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    falciparum strains of step (i) and step (ii);    wherein a decrease in the functional activity of the Plasmodium    falciparum strain of step (ii) relative to the Plasmodium falciparum    strain of step (i) is indicative of the presence of a functionally    inhibitory antibody in said sample.

In another preferred embodiment, there is provided a method of detectingthe presence of a functionally inhibitory antibody in a biologicalsample, which antibody is directed to Plasmodium falciparum MSP-1₁₉,said method comprising:

-   (i) contacting a Plasmodium falciparum schizont of strain D10 with    said sample for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) contacting a Plasmodium falciparum schizont of strain    D10-PcMEGF with said sample for a time and under conditions    sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    falciparum strains of step (i) and step (ii);    wherein a decrease in the functional activity of the Plasmodium    falciparum strain of step (ii) relative to the Plasmodium falciparum    strain of step (i) is indicative of the presence of a functionally    inhibitory antibody in said sample.

To the extent that the method of the present invention is performed invivo, the person of skill in the art must additionally giveconsideration to the strain/species of pathogen which is to beintroduced to the selected host animal. For example, the in vitro modelexemplified herein utilises P. falciparum strains which express eitherthe native form of P. falciparum MSP-1₁₉ or all or part of thehomologous and epitopically divergent P. chabaudi MSP-1₁₉ domain.However, the use of these strains of P. falciparum could only beutilised to perform an in vivo screening assay where the host animalwhich is utilised is one which P. falciparum could colonise. That is,many pathogens demonstrate species specificity. Accordingly, whereas itmay be feasible to directly utilise these specific P. falciparum strainsin some host animals, such as primates, it would not be possible to usethem with a rodent animal model. Accordingly, where it is desired toperform the in vivo screening method described herein, the person ofskill in the art must select, for use, a strain of pathogen which isable to infect the host animal of interest.

For example, and in keeping with the embodiments exemplified herein, anin vivo murine screening assay which is directed to identifying thepresence of functionally inhibitory antibodies directed to P. falciparumMSP-1₁₉ could be performed utilising the P. chabaudi or the P. bergheispecies. These species are both known to colonise mice. In this regard,the “epitopically native” pathogen could be achieved be engineering a P.chabaudi or P. berghei parasite such that it expresses the P. falciparumMSP-1 ₁₉ domain. The “epitopically distinct” pathogen could be provided,for example, in the form of the wild type P. chabaudi or P. bergheiwhich express the murine homolog of the P. falciparum MSP-1₁₉ domain,which form is not recognised by antibodies directed to the P. falciparumform of MSP-1₁₉. In light of the teachings herein, it is within theskill of the person of skill in the art to select, for both in vitro andin vivo application, appropriate species/strains of pathogen for use.Accordingly, it should be understood that in relation to one or both ofthe pathogens expressing the native and epitopically distinct form ofthe antigen in issue, the pathogen species from which the antigen isderived need not necessarily correlate with the species of the pathogenwhich is expressing that antigen. That is, all or some of the pathogenswhich are utilised in accordance with this method may be geneticallyaltered chimaeras. It should also be understood that the functionallyinhibitory antibody which forms the subject of analysis may be one whichwas generated in the mice (for example as a result of the testing of theimmunogenicity of a vaccine) or it may have been administered to themice before, after or together with the pathogen strain (for examplewhere one might be seeking to test in vivo the antibody load present ina human serum sample).

Accordingly, in yet another embodiment there is provided a method ofdetecting the presence of a functionally inhibitory antibody in apopulation of mice, which antibody is directed to Plasmodium falciparumMSP-1 and which method is performed in vivo in said mice, said methodcomprising:

-   (i) introducing to at least one of said mice a wild-type Plasmodium    berghei for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) introducing to at least one of said mice, other than the mosue    of step (i), a Plasmodium berghei strain, which strain expresses the    Plasmodium falciparum MSP-1 block 17C-terminal domain, for a time    and under conditions sufficient to facilitate immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodia of    step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    berghei of step (ii) relative to the Plasmodium berghei of step (i)    is indicative of the presence of said functionally inhibitory    antibody in said mice.

Most preferably, there is provided a method of detecting the presence ofa functionally inhibitory antibody in a population of mice, whichantibody is directed to Plasmodium falciparum MSP-1₁₉ and which methodis performed in vivo in said mice, said method comprising:

-   (i) introducing to at least one of said mice a wild-type Plasmodium    berghei for a time and under conditions sufficient to facilitate    immunointeraction;-   (ii) introducing to at least one of said mice, other than the mouse    of step (i), a Plasmodium berghei schizont of the strain Pb-PfM19    for a time and under conditions sufficient to facilitate    immunointeraction;-   (iii) assessing the level of functional activity of the Plasmodium    berghei strains of step (i) and step (ii)    wherein a decrease in the functional activity of the Plasmodium    berghei strain of step (ii) relative to the Plasmodium berghei    strain of step (i) is indicative of the presence of said    functionally inhibitory antibody in said mouse.

In further aspects of this embodiment said MSP-1 antigen isalternatively the block 2 N-terminal domain, AMA-1, MSP-2, MSP-3, MSP-4,MSP-5, RAP-2, RAP-1, EBA-175 or CSP.

Reference to “biological sample” should be understood as a reference toany sample of biological material derived from an animal such as, butnot limited to, mucus, biopsy specimens, fluid which has been instructedinto the body of animal and subsequently removed such as, for example,the saline solution extracted from the lung following lung lavage,serum, plasma or in vitro derived biological sample such as ascitesfluid or tissue culture supernatant. To the extent that the method ofthe invention is performed in an in vivo model, the biological samplemay be a sample, as detailed above, which is introduced to the animalmodel. Alternatively, where the animal model itself has undergone theinduction of the immune response which is to be analysed in that animal,by virtue of the in vivo analysis method disclosed herein, it should beunderstood that the animal itself falls within the scope of the phrase“biological sample”. The biological sample which is tested according tothe method of the present invention may be tested directly or mayrequire some form of treatment prior to testing. For example, a biopsysample may require homogenisation prior to testing. Further, to theextent that the biological sample is not in a liquid form (for exampleit may be a solid, semi-solid or a dehydrated liquid sample), it mayrequire the addition of a reagent, such as a buffer, to mobilise thesample prior to application of the method of the invention. This samplemay also be treated in terms of undergoing a partial purification step,viral inactivation step or other form of pre-treatment.

The person of skill in the art would understand that steps (i) and (ii)can be performed in either order or simultaneously, the objective ofthese steps being to provide the framework within which a relativeanalysis of the functional readout of step (iii) is facilitated. In thisregard, it should be understood that steps (i) and (ii) need not even beperformed at substantially the same time. For example, in someinstances, which will be obvious to the person of skill in the art,steps (i) and (ii) may be performed days, or even weeks, apart with theresults subsequently analysed relative to one another. Further, it isfeasible that results obtained in relation to step (i) or step (ii)could be utilised as a standard set of control results, thereby enablingthe person of skill in the art to perform, in some suitablecircumstances which would be obvious to the person of skill in the art,only one of step (i) or (ii), subsequently to which the results obtainedthereon are analysed relative to the previously obtained “standard”result.

The method of the present invention is predicated on facilitating theimmunointeraction of an antibody with an antigen. By “immunointeraction”is meant that interaction, binding or other form of association of theantibody of interest with the antigen of interest occurs. It would bewell known to those skilled in the art as to how this could be achievedat either the in vitro or in vivo levels.

Determining the nature of the functional activity which should form thebasis of assessment in relation to any given antigen orimmunointeractive molecule would be determinable by the person of skillin the art based on the common general knowledge. It should beunderstood that assessment of the “level” of functional activity isintended to encompass assessment of the nature or occurrence of aparticular functional activity. Means of assessing the level offunctional activity of the pathogen would be well know to the person ofskill in the art and could be achieved by any convenient means. Further,it should be understood that the method of the present invention can beadapted to screen for the subject antibodies at the qualitative and/orthe quantitative levels.

In the method of the invention exemplified herein, the antibodies whichare screened for are human antibodies directed to the P. falciparumMSP-1₁₉, which antibodies prevent invasiveness of the P. falciparummerozoite. In accordance with this objective, one embodiment of theinvention is directed to screening human serum samples in an in vitroassay where ring-stage parasites (D10 parasites being the parasiticstrain which expresses the native form of MSP-1₁₉ and D10-PcM3′ orD10-PcMEGF which express some or all of the P. chabaudi MSP-1₁₉antigens) are synchronised and allowed to mature through to thetrophozoite/schizont stage. These mature parasites are then co-culturedwith red blood cells and the serum sample of interest. These culturesare incubated for a time and under conditions sufficient to allow forschizont rupture, merozoite invasion and antibody binding. Theassessment of functional activity of the Plasmodium falciparum strainstested herein is assessed by three mechanisms as follows:

-   (i) Microscopy analysis: Smears are made of the cultured parasites    and the number of ring-stage parasites per red blood cells is    determined. The mean parasitemia can then be calculated and    expressed as a percentage of the mean parasitemia observed in    parallel cultures.-   (ii) [³H]hypoxanthine uptake assay: Following culture of the    parasites, the culture medium is removed and replaced with    hypoxanthine-free medium supplemented with [³H]hypoxanthine.    Following a further 24 hours of culture the mature parasites are    frozen and thawed in order to effect lysis of the infected red blood    cells. Samples are then transferred to glass fibre filters via a    cell harvester and quantitated using a scintillation counter.-   (iii) Co-cultivation assays: In these assays the epitopically    native P. falciparum strain and the epitopically distinct P.    falciparum strain are co-cultured subsequently to having been    synchronised at ring stage and allowed to mature through to the    trophozoite/schizont stage. They are cultured together at an equal    ratio in the presence of serum. Parasites are smeared at the    trophozoite/schizont stage and assessed by indirect    immunofluorescence.-   (iv) FACS detection: For example cultures are set up as for    microscopy analysis. The parasites are allowed to mature a further    24 hours (ie. 48 hours after set-up) and are then labelled with    hydroethidine (HE). HE is incorporated into the DNA of viable    parasites only and can be detected by flow cytometry. HE is added to    parasites, incubated at 37° C. in the dark for 20 minutes, diluted    with buffer and pelleted. Pellets are resuspended for FACS.

The development of a method of detecting functionally inhibitoryimmunointeractive molecules now facilitates its application to a rangeof situation including, but not limited to:

-   (i) predicting the immune status of individuals who have been    previously infected with a pathogen;-   (ii) predicting the immune status of individuals vaccinated with    vaccines designed to administer a specific antigen. This is    particularly valuable in relation to clinical trials;-   (iii) determining the relative contribution of an antibody of    specific immunoreactivity to the total inhibitory antibody elicited    by combination vaccines which include two or more antigens, one of    which is the antigen to which the subject antibody is directed.-   (iv) the assessment of human vaccines in a mouse model system. This    is particularly important with respect to assessing the efficacy of    different forms of an antigen which are proposed to be utilised in a    vaccine (eg. antigens which have been prepared utilising different    methodologies) and assessing vaccine potency (which for some    vaccines reduces over time when stored);-   (v) assessment of the protective potential of an antibody of    interest which is present in human serum (for example, of infected    or vaccinated individuals). This requires passive transfer of human    antibodies into mice which are subsequently infected with the    epitopically distinct pathogen in issue, as detailed hereinbefore.-   (vi) determination of the importance of functionally inhibitory    antibodies to clinical protection. This is of particular importance    since it will reveal much about the mechanism of protection which    occurs in immune individuals.

Accordingly, in another aspect the present invention is directed to amethod of assessing the nature of an immune response to an antigen inaccordance with the methods defined hereinbefore. The term “nature”should be understood in its broadest sense as a reference to any one ormore qualitative and/or quantitative aspects of an immune response. Asdetailed above, this provides a means of assessing an immune response toa pathogen in accordance with points (i)-(vi), above.

In yet another aspect, the present invention extends to the pathogensdefined herein.

Accordingly, yet another aspect of the present invention is directed toan isolated pathogen, which pathogen expresses a non-wild-type form ofone or more antigens derived from said pathogen.

More particularly, the present invention provides an isolated malariapathogen, which pathogen expresses a non-wild-type form of one or moreantigens derived from said pathogen.

It should be understood that reference to the terms “pathogen” and“malaria” have the same meaning as hereinbefore defined. Similarly, thephrase “antigen(s) derived from said pathogen” should be understood tohave the same meaning as the previously defined phrase “pathogen derivedantigen”. Also, as detailed hereinbefore, the antigen may be one whichis permanently or transiently expressed in either a constitutive orinducible manner. This may largely depend on the life-cycle stage of thepathogen at any given point in time.

Reference to a “non-wild-type” form of an antigen should be understoodas a reference to the form of the subject antigen which differs from theform expressed by the wild-type form of the pathogen. In this regard,the non-wild-type form will generally differ from the wild-type form byvirtue of the amino acid sequence of the subject antigen. However, thepresent invention is not limited in this regard and any other form ofchange which would render an antigen “non-wild-type” is encompassed inthis definition. In the context of the present invention, thenon-wild-type form of the antigen will generally correspond to ahomologous form of the antigen. For example, to the extent that themethod of the present invention is applied to detecting the generationof antibodies in a mouse, directed to a human pathogen, one wouldutilise a form of the pathogen which can colonise and replicate in mice(since the assay is to be performed in mice) but wherein the viabilityin those mice of the wild-type form of the pathogen is analysed relativeto a murine form of the pathogen which has been engineered to expressthe human version of the antigen to which the antibodies have beenraised. In this regard, and as detailed hereinbefore, the murine form ofthe parasite which expresses the human homolog of the antigen to whichantibodies may have been raised corresponds to the form of pathogenexpressing an “epitopically native form” of the antigen since this isthe form of antigen against which it was desired to raise antibodies.The wild-type form of the pathogen should be understood to express the“epitopically distinct form” of the antigen since it expresses a form ofantigen against which the antibodies were not directed. Accordingly,reference to an antigen being “epitopically distinct” versus“epitopically native” is assessed relative to the form of antigenagainst which the presence of the functionally inhibitory antibody isbeing assessed. It should also be understood, however, that to theextent that the use of “wild-type” forms of pathogens are defined in themethods hereinbefore, a pathogen will satisfy that this definitionprovided it is “immunologically” wild-type. That is, that the antigenregion in issue is not immunogenic in the species to which it isadministered. Accordingly, some small changes to the subject “antigen”region may not change its immunogenicity and therefore render thosepathogens effectively useful as “wild-type” pathogens.

Preferably, the present invention provides an isolated Plasmodium, whichPlasmodium expresses a non-wild-type form of MSP-1.

More preferably, said MSP-1 is the block 17 C-terminal domain or theblock 2 N-terminal domain of MSP-1.

Most preferably, said Plasmodium is Plasmodium berghei expressing thePlasmodium falciparum form of the MSP-1₁₉ antigen.

Even more preferably, said Plasmodium berghei is the Pb-PfM19 strain.

In another embodiment, the present invention provides an isolatedPlasmodium pathogen expressing a non-wild-type form of one or moreantigens derived from said pathogen, which antigens are selected fromthe list of:

-   (i) the apical membrane domain (AMA-1)-   (ii) merozoite surface protein 2, 3, 4 and/or 5 (MSP-2, MSP-3, MSP-4    and/or MSP-5)-   (iii) rhoptry associated protein 2 (RAP-2)-   (iv) erythrocyte binding antigens (EBA-175)-   (v) circumsprozoite antigen (CSP)

In accordance with this preferred embodiment, still more preferably thesubject malaria pathogen is a Plasmodium pathogen and still morepreferably a Plasmodium falciparum pathogen, Plasmodium berghei pathogenand/or Plasmodium chabaudi pathogen.

In yet another aspect, the present invention extends to the pathogensdefined herein when used in accordance with the method of the presentinvention.

Further features of the present invention are more fully described inthe following non-limiting Examples.

EXAMPLE 1 Antibodies Against Merezoite Surface Protein (MSP)-1₁₉ are aMajor Component of the Invasion Inhibitory Response in IndividualsImmune to Malaria

Materials And Methods

Plasmids

Construction of the plasmids pFfM3′ and pPcM3′ has been describedpreviously (O'Donnell, R. A. et al. 2000 supra). The plasmid pPcMEGF wasconstructed by the insertion of a 1,200-bp XhoI fragment into the uniqueXhoI site of a plasmid pHC2 (Triglia, T., Healer, J., Caruana, S. R.,Hodder, A. N., Anders, R. F., Crabb, B. S. and Cowman, A. F. (2000) Mol.Microbiol. 38:706-718). This target fragment comprises a 900-bp internalregion of the P. falciparum MSP-1 gene fused in frame to the MSP-1₁₉region of P. chabaudi. The fragment was generated by PCR amplificationfrom P. falciparum (D10) and P. chabaudi (adami DS) genomic DNA (gDNA)using the oligonucleotide pairs Pf#1 5′-ATTTCTCGAGAATCCGAAGATAATGACG-3′(<400>1), PfEGF-R5′-GAAACATCCAGCATTTTCTGGAAGTTTGTTCCTATGCATTGGTGTTGTGAAATG-3′ (<400>2).The resulting amplicons were sewn together via PCR for insertion intopHC2. The XhoI sites are shown in bold.

Parasite Culture and Transfection Procedures

P. falciparum line D10 was cultivated and synchronised as per standardprocedures (Larnbros, C. and Vanderberg, J. P. (1979) J. Parasitol.65:418-420; Trager, W. et al. 1976). Ring-stage parasites (˜5%parasitemia) were transfected with 50-100 μg of CsCl-purified plasmidDNA as described previously (Crabb, B. S. and Cowman, A. F. (1996) Proc.Natl. Acad. Sci. USA. 93:7289-7294; Crabb, B. S., Triglia, T.,Waterkeyn, J. F. and cowman, A. F. (1997) Mol. Biochem. Parasitol.90:131-144) but using the electroporation conditions as described byFidock and Wellems (Fidock, D. A. and Wellems, T. E. (1997) Proc. Natl.Acad. Sci. USA. 94: 10931-10936). After transfection and initialselection using 0.1 μM pyrimethanine for ˜4 weeks, parasites weresubjected to repeated cycles of 1 μM pyrimethamine for 3 weeks proceededby removal of the drug for 3-4 weeks. gDNA was extracted from mixedtrophozoit/schizont stage parasites as described previously (Coppel, R.L. and Biano, A. E., Culvenor, J. G., Crewther, P. E., Brown, G. V.,Anders, R. F. and Kemp, D. J. (1987) Mol. Biochem. Parasitol. 25:73-81),and Southern blot analysis was carried out using standard procedures(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York).

Western Blot Analysis

Parasite proteins were obtained from extracted enriched schizont ormerozoite preparations and separated using 7.5 and 12% SDS-PAGEnonreducing gels, respectively, and transferred to PVDF membranes(Amersham Pharmacia Biotech). membranes were probed with either mouseascitic fluid containing 4H9/19, a monoclonal antibody specific for P.falciparum MSP-1₁₉ (Cooper, J. A. et al. 1992 supra), diluted 1:80,000or rabbit *PcM19 polyclonal antibodies diluted 1:2,500 that are specificfor P. chabaudi MSP-1₁₉ (O'Donnell, R. A. et al. 2000 supra).Horseradish peroxidase-conjugated rabbit anti-mouse (Dako) or sheepanti-rabbit (Silenus) Igs were used for detection, and bands werevisualised by enhanced chemiluminescence (NEN Life Science Products).

Indirect Immunofluorescence

For indirect immunofluorescence assay (FA), D10-PfM3′ and D10-PcMEGFschizont-stage parasites were incubated with a mixture of 4H9/19 asciticfluid and *PcM19 sera diluted 1:4,000 and 1:1,000, respectively. Afterincubation in the presence of a mixture of FITC-conjugated sheepanti-mouse and rhodamine-conjugated goat anti-rabbit Igs (Dako), bothdiluted 1:150, parasites were visualised by fluorescence microscopy. Thesame fields were photographed using filters to detect the FITC orrhodamine fluorochromes.

Sera

The Papua New Guinean sera used were collected in the Madang Provincefrom adults living in and around Madang town in 1980-82 (denoted PNG-Msera) and from adults currently living on Bagabag Island (denoted PNG-Bsera). both locations have high prevalence rates of P. falciparum (overall rates of 25.7 and 24%, respectively, with the highest rates observedin 1-9 year-old children in both communities) that are indicative ofintense transmission (Cattani, J. A., Tulloch, J. L., Vrbova, H.,Jolley, D., Gibson, F. D., Moir, J. S., Heywood, P. F., Alpers, M. P.,Stevenson, A. and Clancy, R. (1986) Am. J. Trop. Med. Hyg. 35:3-15).Transmission in these localities is perennial with similar rates in thewet and dry seasons.

To generate P. chabaudi immune mouse sera (Pc immune), six 7-week-oldC57BL/6 male mice were injected intraperitoneally with 5×10³ P. chabaudi(adami DS)-infected RBCs and rechallenged at 3 weeks with the same dose.At weeks 7 and 21, mice were administered a higher challenge of 10⁴ P.chabaudi-infected RBCs before serum collection at week 24.

MSP-1₁₉ Glutathione S Transferase Fusion Proteins

The DNA sequence corresponding to the MSP-1₁₉ fragment lacking theglycosylphosphatidylinositol anchor sequence (amino acids Asn 1631-Ser1723, according to reference 27) was amplified from P. falciparum D10 orHB3 gDNA (which contains the MAD20 or K1 MSP-1₁₉ alleles, respectively;reference 27) using the oligonucleotides: PfM19f5′-CGCGGATCCAACATTTCACAACACCAATGCG-3′ (<400>3) and PfM19r5′-GGAAGATCTTAACTGCAGAAAATACCATCGAAAAG-3′ (<400>4). The resulting PCRproducts were ligated into the BamHI site of pGEX-4T-1, expressed asglutathione S transferase (GST) fusion proteins in Escherichia coli(Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40) and purifiedusing glutathine-sepharose as described by the manufacturer (AmershamPharmacia Biotech). GST alone was produced using the pGEX-4T-1 plasmid.

ELISA

Antibodies reacting with recombinant P. falciparum MSP-1₁₉ and P.chabaudi MSO-1₁₉ were detected by ELISA. Microtitre plates (Dynex) werecoated overnight at 4° C. with 0.5 μg/ml recombinant protein diluted incarbonate buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃, pH 9.6). Plates werewashed three times with PBS containing 0.05% Tween 20 (PBST), blockedfor 2 hours at 37° C. with PBST containing 10 mg/ml BSA, and washedagain. Sera diluted in PBST containing 10 mg/ml BSA, and washed again.Sera diluted in PBST containing 5 mg/ml BSA was then added to the plates(50 μl), which were then incubated further for 1 hour at 37° C. Afterwashing with PBST, horseradish peroxidase-conjugated sheep anti-humanIgG (1:4,000; Silenus), rabbit anti-mouse IgG (1:5,000), or sheepanti-rabbit IgG (1:2,500; Dako) were added to the plates, incubated for1 hour at 37° C. and, after washing with PBST, developed with H₂O₂ and3,3′, 5,5′-tetramethylbenzidine dihydrochloride for 10 min at roomtemperature. The reaction was stopped by the addition of 20 μl 2.5 MH₂SO₄ and plates were read at 450 nm. All human and mouse sera weretested in duplicate at three dilutions (1:1,000, 1:3,200, and 1:10,000)against GST-PfM19, GST-PcM19, and GST alone. The mean optical density(OD) value derived from GST alone was subtracted from the mean ODobtained for each GST fusion protein. Values at a 1:3,200 dilution wereconsidered most likely to be on the slope of a titration curve, hence,these values are represented here.

Inhibition of Invasion Assays

Ring-stage parasites were synchronised by sorbitol lysis twice at 4 hourintervals and then allowed to mature through to trophozoite/schizontstages. the purified parasites were adjusted to 4% hematocrit with0.5-2% infected RBCs and aliquots of 50 μl placed into the wells of a96-well tray. An equal volume of serum, prediluted 1:10 in culturemedia, was added (resultant hematocrit of 2%) and cultures incubated for˜26 hours to allow for schizont rupture and merozoite invasion. Notethat when two parasite lines were being compared the same batch ofprediluted serum was added to each line. For the microscopy analysis,smears were made of the duplicate wells, stained with Giemsa, and thenumber of ring-stage parasites per 500 RBCs were determined for eachwell. The mean parasitema from duplicate wells was calculated and thiswas expressed as a percentage of the mean parasitemia observed inparallel cultures of each parasite line in the presence of pooled humannonimmune sera (HNIS). For the [³H]hypoxanthine uptake assay, media wasremoved from triplicate wells at ˜24 hours after cultivation andreplaced with hypoxanthine-free media supplemented with [³H]hypoxanthine(10 μCi/ml). A further 24 hours later, cultures of mature parasites werefrozen and thawed to lyse infected RBCs. Samples were transferred toglass fiber filters via a cell harvester and quantitated using ascintillation counter. Statistical analysis for address whether the meaninvasion rates of these sera were the same between two lines wasperformed using a two-sample Student's t test assuming unequalvariances.

Co-cultivation Assays

For co-cultivation, D10-PfM3′ and D10-PcMEGF ring-stage parasites weredoubly synchronised as described above and then cultured together at anequal ratio in the presence of pooled sera. Sera was pooled on the basisof either appearing to contain significant proportions of anti-MSP-1₁₉inhibitory antibodies (pool 2) or having a less inhibitory effectbetween the two parasite lines (pool 1). The pools included thefollowing sera: (PGN-B pool 1) 8, 247, 332, and 962; (PNG-B pool 2) 413,604, 614, and 954; (Pc-immune pool 1) 2 and 4; and (Pc-immune pool 2) 1,3, 5, and 6. Additional controls included a HNIS pool and a *PcM19rabbit serum. Parasites were smeared at the trophozoite/schizont stage(ie., every 2 days beginning at day 1) and assessed by indirect IFAusing a mixture of 4H9/19 and *PcM19 as described above. For each of thetriplicate slides, FITC (DIO-PfM3′) and rhodamine (D19-PcMEGF) wereobserved and counted in 16 individual fields that each contained atleast 10 mature (pigmented) parasites. In total, between 400 and 1,400mature stage parasites were counted for each co-cultivation sample.

Results

Replacement of Complete EGF Domains of P. falciparum MSP-1₁₉ with thosefrom P. chabaudi

The aim of this study was to generate a P. falciparum line thatpossesses an antigenically distinct MSP-1₁₉ domain and to investigatewhether this line differs from parental parasites in its susceptibilityto inhibition by sera from malaria-immune individuals. We have describedpreviously the construction of a parasite line D10-PcM3′ which expressesan MSP-1 chimera in place of the endogenous molecule (O'Donnell, R. A.et al. 2000 supra).

This chimera incorporates ˜¾ of the two EGF-like domains that compriseMSP-1₁₉ from the divergent rodent malaria P. chabaudi (FIG. 1A). We alsoconstructed a control transfectant D10-PfM3′ which expresses endogenousMSP-1 (FIG. 1A; reference 11). These transfected lines displayed noobservable phenotypic differences to parental D10 parasites revealingthat the function of most of MSP-1₁₉ is conserved across divergentPlasmodium species. Here we describe transfection of a plasmid, pPcMEGF,designed to replace the entire EGF domains from MSP-1₁₉ with those fromP. chabaudi (FIG. 1). Upon transfection and drug cycling, pPcMEGF wasshown to have integrated into the MSP-1 gene. The transfectedpopulation, D10-PMEGF, was cloned and two randomly selected clones(D10-PcMGF.1 and D10-PcMEGF.2) were analyzed further. Southern blotanalysis showed the plasmid had integrated into the target site throughthe expected recombination event replacing the entire endogenous P.falciparum MSP-1₁₉ EGF domains with those from P. chabaudi. This linewould be distinguished from both D10-PfM3′ and D10-PcM3′ by restrictionendonuclease digestion with XbaI (FIG. 1, B and C).

To determine if the chimeric MSP-1 was expressed in D10-PcMEGFparasites, extracts of mature schizonts and free merozoites wereexamined by immunoblot analysis (FIG. 2A). Bands corresponding toendogenous full-length MSP-1 (˜200 kD) and MSP-1₁₉ (˜18 kD) weredetected in parental D10 schizonts and merozoites, respectively, usingthe P. falciparum-specific antibody 4H9/19 (Cooper, J. A. et al. 1992supra). No reactive bands were observed in extracts from the twoD10-PcMGF clones. Conversely, when replicate immunoblots were probedwith a rabbit antiserum specific for P. chabaudi MSP-1₁₉ (*PcM 9;reference 11), species corresponding to both forms of MSP-1 wereobserved in the D10-PcMEGF extracts but not in parental D 10 (FIG. 2A).The larger band (40 kD) in the merozoite samples is consistent with thepresence of the primary MSP-1 processing product, MSP-1₄₂. Thelocalisation of the MSP-1 chimera was assessed by an IFA (FIG. 2B).D10-PfM3′ and D10-PcMEGF parasites were incubated with a mixture ofmouse 4H9/19 and rabbit *PcM19 antibodies followed by FITC-labelledanti-mouse (to detect endogenous MSP-1) and rhodamine-labelledanti-rabbit (to detect the MSP-1 chimera) IgG. “Grape-like” fluorescencewas observed in both lines indicative of merozoite surface labelling.D10-PcMEGF parasites showed only rhodamine fluorescence supporting theabsence of endogenous MSP-1₁₉ expression in this line. Fluorescence wasalso observed in ring-stage parasites indicating that the P. chabaudiMSP-1₁₉ domain is carried into the newly invaded RBCs in D10-PcMEGFparasites as has been described for P. falciparum MSP-1₁₉ (data notshow; references 11 and 12).

To ensure that the chimeric MSP-1 was functional, an in vitro inhibitionof invasion assay was carried out (FIG. 2C). Mature stage parasites fromparental D10, D10-PcM3′, and two clones from D10-PcMEGF were incubatedin the presence of *PcM19 IgG. These antibodies specifically inhibitedRBC invasion of D10-PcMEGF and D10-PcM3′ parasites in a dose-dependentmanner but had no effect on parental D10. These results are consistentwith the correct expression, processing, localisation and functioning ofthe expected hybrid MSP-1 molecule in D10-PcMEGF parasites. This alsoreveals that the complete EGF domains of MSP-1₁₉ are functionallyconserved across distantly related Plasmodium species.

Invasion-inhibition of Transfected P. falciparum Parasites by ImmuneSera Reveals an Important Role for MSP-1₁₉ specific Antibodies

The availability of parasite lines that are identical except for thepresence of antigenially distinct MSP-1₁₉ domains provided a uniqueopportunity to address the relative importance of MSP-1₁₉ antibodies toinvasion-inhibition by immune sera. Sets of human sera were obtainedfrom adults in two malaria endemic areas in Papua New Guinea that haveintense transmission rates of P. falciparum (PNG-M and PNG-B). Themajority, if not all, of these individuals are likely to be clinicallyimmune to P. falciparum malaria. Sera from six C57BL/6 mice that hadbeen repeatedly infected with P. chabaudi were also generated (Pc-immunesera). To determine the presence and the specificity of MSP-1₁₉antibodies, each human and mouse serum was tested in ELISA againstrecombinant forms of P. falciparum MSP-1₁₉ (GST-PfM19) and P. chabaudiMSP-1₁₉ (GST-PcM19). All PNG-B sera (47/47) and most PNG-M sera (27/33)reacted against GST-PfM19 while only five human sera (all from PNG-B)showed detectable cross-reactivity with GST-PcM19 (Table 1). The OD₄₅₀values against GST-PcM19 of these five cross-reactive PNG-B sera rangedfrom 0.277 to 0.900 (mean=0.451). The remaining 42 PNG-B serum sampleshad OD₄₅₀ values against GST-PcM19 below 0.113.

The six mouse sera showed no reactivity to GST-PFM19 but each showedstrong reactivity with GST-PcM19. These results reveal that MSP-1₁₉antibodies were generated in response to infection with either P.falciparum or P. chabaudi and that these were mostly highly specific forthe homologous MSP-1₁₉ domain. The P. falciparum MSP-1₁₉ sequence ofGST-PFM19 represented the “MAD20” allele; however, each serum was alsotested in parallel with a GST-MSP-1₁₉ fusion protein comprising“KI/Wellcome” allelic sequence (Miller, L. H., Roberts, T., Shahabuddin,M. and McCutchan, T. F. (1993) Mol. Biochem. Parasitol. 59:1-14; Tanabe,K., Mackay, M., Goman, M. and Scaife, J. G. (1987) J. Mol. Biol.195:273-287). The OD readings against this fusion protein were verysimilar to those obtained for the “MAD20” allele across all PNG-B andPNG-M sera (R²=0.914). This cross-reactivity between alleles isconsistent with the finding of others (Egan, A. F., Chappel, J. A.,Burghaus, P. A., Morris, J. S., McBride, J. S., Holder, A. A., Kaslow,D. C. and Riley, E. M. (1995) Infect. Immun. 63:456-466).

Preliminary experiments in our laboratory had indicated that D10-PcMEGFwere relatively resistant to inhibition by human sera frommalaria-immune individuals. To explore this more thoroughly, all PNG-M,PNG-B, and Pc-immune mouse sera were assessed for their ability toinhibit invasion of D10 and D10-PcMEGF merozoites in a microscopy-basedinvasion inhibition assay. All sera were tested in the one assay withthe same parasite preparations (assay 1; FIG. 3A). PNG-B sera wererelatively effective at inhibiting invasion of parental D10 parasiteswith a mean invasion of 26.7%. PNG-M sera were generally less inhibitoryof D10 parasites (43.1%). The difference between PNG-B and PNG-M sera,both in invasion-inhibition and total MSP-1₁₉ antibodies (Table I), maysimply reflect a loss of potency of PNG-M sera over relatively long-termcryopreservation period (˜20 years) although this was not exploredfurther.

Strikingly, we found that both PNG-B and PNG-M sera were generally muchless effective at inhibiting the invasion of 10-PcMEGF merozoites (FIG.3A). Here, mean invasion rates of 52.3 and 66.4% were obtained for PNG-Band PNG-M, respectively, which in both cases was ˜25% higher than thatobtained for D10. In contrast, the Pc-immune sera was more effective atinhibiting D10-PcMEGF (mean invasion rate 57.5%) than parental D10 (meaninvasion 73.5%). In each case, the difference in the mean invasion ratewas either significant or highly significant (FIG. 3A).

In an attempt to independently confirm these result, the inhibitorypotential of these sera was tested by a different assay that utilizes[³H]hypoxanthine uptake as a measure of parasite growth (assay 2; FIG.3B). In this assay, D10-PfM3′ was used as the parental control insteadof D10 and again all sera were tested together in the one assay. Theresults were similar ot those obtained in assay 1 with D10-PfM3′parasites more susceptible than D10-PcMEGF to inhibition by PNGOM andPNGOB sera. Again, as in FIG. 3A, D10-PcMEGF parasites were moresusceptible than D10-PfM3′ to inhibition by Pc-immune sera. In eachcase, the difference in the mean invasion rates was highly significant.These results are consistent with a major role for MSP-1₁₉ antibodies ininvasion/growth inhibition by malaria immune sera.

FIG. 4 shows inhibition results (from assay 2) that are representativeof the data obtained for individual sera. Although some individual humansera did not appear to contain high levels of P. falciparumMSP-1₁₉-specific inhibitory antibodies (eg. 938, 961, and 1,057), amajor proportion of the invasion-inhibitory component of other sampleswas directed against MSP-1₁₉ (eg. 406, 604, 724). Most human samples(59/80) showed some level of P. falciparum MSP-1₁₉-specfic inhibitoryantibodies in either assay 1 or 2. All Pc-immune sera had detectablelevels of P. chabaudi MSP-1₉-specific inhibitor antibodies in eitherassay 1 or 2. Results for the two control sera used in assay 2 are alsoshown (FIG. 4). the first was a polyclonal rabbit anti-P. falciparumAMA-1 IgG (Hodder, A. N., Crewther, P. E. and Anders, R. F. (2001)Infect. Immun. 69:3286-3294) used at a concentration of 250 μg/ml andthe second was *PcM19 purified IgG used at a concentration of 750 μg/ml.both lines were equally susceptible to inhibition by *AMA-1 IgG, whereasonly D10-PcMEGF was inhibited with *PcM19.

We also examined if there was any relationship between the amounts ofMSP-1₁₉-specific invasion-inhibitory antibody and total MSP-1₁₉ IgG.MSP-1₁₉ invasion-inhibitory antibody in each human serum was calculatedfrom microscopy-based assay by subtracting the percentage of invasionfor D10-PfM3′ from the value obtained with D10-Pc-MEGF. These valuesshowed no correlation with the OD₄₅₀ readings obtained for each serumagainst GST-PfM19 antigen (R²=0.013 and 0.0003 for PNG-M and PNG-B sera,respectively). However, it should be noted that four of the six PNG serathat were negative for GST-PfM19 antibodies (at a 1:3,200 dilution) inELISA (Table I) also showed no detectable levels of MSP-1₁₉-specificinvasion-inhibitory antibodies. The amount of P. chabaudiMSP-1₁₉-specific inhibitory antibody present in individual Pc-immunesera also showed no relationship to total P. chabaudi MSP-1₁₉-specificIgG (R²=0.0048).

Co-cultivation Assays in the Presence of Sera from Immune IndividualsSupport a Major role for MSP-1₁₉ Antibodies in Growth Inhibition

As an alternative means of addressing the specificity of the inhibitoryantibodies in immune sera for MSP-1₁₉, D10-PfM3′ and D10-PcMEGFparasites were co-cultivated at an equal ratio in the presence of pooledsera. Several individual sera were pooled on the basis of the amount ofanti-MSP-1₁₉-inhibitory antibody determined by the inhibition assaysdescribed above. Those with lower levels of MSP-1₁₉-specific inhibitoryantibody comprised pool 1 while those with more apparentMSP-1₁₉-inhibitory antibody comprised pool 2. Parasites were detected byindirect IFA using a mixture of 4H9/19 and *PcM19 to detect D10-PfM3′and D10-PcMEGF, respectively. The insert for FIG. 5 shows a typicalfield after incubation with pooled HNIS showing similar numbers ofD10-PfM3′ (green) and D10-PcMEGF (red) parasites and illustrates theease with which the two different lines were visualised in the mixedculture.

Red and green fluorescent parasites were counted after 1 and 5 days ofco-cultivation in the presence of the different pooled sera. After 1 dayof culture, where parasites were expected to have matured but notreinvaded fresh RBCs, no change in parasite ratio was observed with anysera. Co-cultivation in the presence of HNIS for 5 days also had noeffect on the ratio of the two parasite lines confirming hat D10-PfM3′and D10-PcMEGF have very similar growth rates (FIG. 5). However,incubation of the parasite mix with *PcM19 or Pc-immune sera had adramatic effect on parasite ratio with the number of D10-PfM3′ parasitesin 3-4-fold excess of D10-PcMEGF. In contrast, incubation withPNG-B-pooled sera had the opposite effect. It is important to note thatthe human and mouse sera pooled on the basis of possessing the mostMSP-1₁₉-inhibitory antibody in the aforementioned assay (FIG. 3) alsoexhibited the greatest growth inhibition here.

EXAMPLE 2 Generation of Recombinant Parasites

Methods

Plasmids

The pHCl plasmid vector has been described (Crabb, B. S. et al. 1997supra). XhoI insers for cloning into this plasmid were amplified fromthe relevant genomic DNA using the following oligonucleotides(restriction endonuclease sites are bolded):Pf#1,5′-ATTTCTCGAGAATCCGAAGATAATGACG-3′ (<400>5);Pf#2,5′-ATTGCTCGAGATCGATGTTTAACATATCTTGGAATTTTTCC-3′ (<400>6); Pf#3,5′-TTTAACTCGAGCATTTTTTAAATGAAACTG-3′ (<400>7);Pf#4,5′-CATCTAGATGTCTGAAACATCCAG-3′ (<400>8);Pc#1,5′-GGATGTTTCAGACATCTAGATGGTAAAG-3′ (<400>9);Pc#2,5′-TCACTCGAGTTAAAATAAATTAAATACAATTAATGTG-3′ (<400>10). To derivethe pAMSPI and pPfM3′ fragments, Pf#1/Pf#2 and Pf#1/Pf#3 were used,respectively. to derive the pPcM3′ insert, amplicons from Pf#1/Pf#4 andPc#1/Pc#2 were first digested with XbaI and ligated. The pPcM3′ vectoris identical to pHCl except that it has a litmus 28 (NEB) backbone.

Parasite Transfection

Plasmids were transfected into P. falciparum parasites (D10 line)essentially as described (Crabb, B. V. et al. 1996 supra). Aftertransfection and initial selection using 0.1 μM pyrimethamine forapproximately 4 weeks, parasites were subjected to cycles of 1 μMpyrimethamine for 3 weeks followed by removal of the drug for 3-4 weeks.To detect homologous integration events, PCR was done on genomic DNAusing a P. falciparum MSP-1 forward primer(5′-GTGAAAATAATAAGAAAGTTAACGAAGC-3′ (<400>11)) located upstream of thetarget sequence together with an HSP863′ reverse primer(5′-GTATATTGGGGTGATGATAAAATGAAAG-3′ (<400>12)). The identity of theseproducts, which are only amplified if homologous integration hasoccurred, were confirmed by nucleotide sequencing.

Generation of P. chabaudi MSP-1₁₉ Antisera

The sequence of MSP-1₁₉ was amplified from P. chabaudi (adami D5) Dnausing the oligonucleotides 5′-CACATACCCTCAATAGCTTT-3′ (<400>13) and5′-GCTGGAAGAACTACAGAATA-3′ (<400>14), and was ligated into pFLAG(Eastman Kodak, Rochester, N.Y., USA) protein was concentrated fromculture supernatants by differential ammonium sulfate precipitation,bound to a Q Sepharose ion exchange column in 25 mM histidine-HCl, pH5.7, and eluted with a NaCl gradient on 0-0.5 M. It was then purifiedsing a second ion exchange chromatographic step on a Biosepra Q HyperDcolumn again with a NaCl gradient of 0-0.5 M in a buffer of 25 mMhistidine-HCl, pH 5.7. To generate αPcM19 antisera, two rabbits (A andB) were inoculated intramuscularly with 100 μg protein in Freund'scomplete adjuvant and were boosted twice with 100 μg protein in Freund'sincomplete adjuvant. Antiserum from rabbit B was used throughout thisstudy, except where indicated otherwise.

Growth Rate and Co-cultivation Assays

Parasites were cultured in the absence of pyrimethamine for at least 1week before these assays. For the growth rate assay, parasites weresynchronised by lysis of ‘non-ring stage’ forms with 5% (w/v) sorbitalin distilled water, at 4-hour intervals, and then plated in duplicate at0.5% parasitemia in medium containing 4% hematocrit. Thin blood smearswere made every 9 hours to court parasites. Fresh media was added daily,and every 48 hours cultures were diluted 1:5 with fresh mediumcontaining 4% hematocrit. For co-cultivation, after doublesynchronisation as described above, D10-PfM3′ and D10-PcM3′ ring-stageparasites were mixed at four different ratios and maintained in mediumcontaining 4% hematocrit. Parasites were smeared at thetrophozoite/schizont stage at day 1 and after two cycles at day 5. Thesesmears were assessed by indirect immunofluorescence assay using amixture of 4H9/19 and αPcM19 antibodies. Assays were done by incubatingschizonts at 2% hematocrit for 23 hours in the presence of purifiedαPcM19 IgG from rabbits A and B. Assays were done in triplicate. Foreach well, the number of ring-stage parasites per 2,000 cells wascalculated. For each slide, parasites labelled with fluoresceinisothiocyanate (FITC; D10-PfM3′) and rhodamine (D10-PcM3′) were countedin about 15 individual fields that each contained at least 20mature-stage parasites. Results are expressed as a ratio of D10-PfM3′ toD10-PcM3′ parasites.

Western Blot Analysis and Indirect Immunofluorescence

Parasite proteins were obtained from extracted enriched schizonts ormerozoites preparations, and separated by 7.5% and 12% SDS-PAGE,respectively, in nonreducing conditions and transferred to PVDFmembranes (Millipore, Bedford, Mass.). These were probed with either4H9/19 antibody, diluted 1:10,000, or αPcM19 antibody, diluted 1:2,000.Parasite extracts were from parental D10, D10-PfM3′ parasites (PfM3′)and the cloned lines from D10-PcM3′ (PcM3′.1 and PcM3′.2). Molecularweight standards were obtained from BioRad (Richmond, Calif.).

For immunofluorescence, D10, D10-PfM3′ (PfM3′) and D10-PcM3′.1 (PcM3′)schizont-stage or ring-stage parasites were incubated with a mixture of4H9/19 and αPcM19 antibodies, each diluted 1:2,000. After incubation inthe presence of a mixture of FITC-conjugated antibody against mouse andrhodamine-conjugated antibody against rabbit immunoglobulins (Dako,Carpinteria, Calif.), both diluted 1:200, parasites were visualised bymicroscopy. The same fields were photographed with bright-field (light)and fluorescence conditions to detect the FITZ or rhodaminefluorochromes.

EXAMPLE 3 A New Rodent Model to Assess Blood-Stage Immunity to thePlasmodium Falciparum Antigen MSP-119 Reveals a Protective Role forInvasion Inhibitory Antibodies

Materials And Methods

Plasmids

To create the pPb-PfM19 replacement plasmid, 1.3 Kb of P. berghei MSP-1targeting sequence was firstly fused in frame to the MSP-1₁₉ region ofP. falciparum upstream from the first cysteine residue of EGF domain 1.This was achieved by PCR amplification of P. berghei ANKA and P.falciparum D10 genomic DNA (gDNA) using the oligonucleotide pairs PbF(5′-CGGGGTACCATCGATAAATACTTTACCTCTGAAGCTGTTCC (<400>15)) and PbR1(5′-TACATGCTTAGGGTCTATACCTAATAAATC (<400>16)), and PbPfF(5′-GGTATAGACCCTAAGCATGTATGCGTAAAAAAACAATGTCCAGAA (<400>17)) and PfR(5′-TGCTCTAGATTAAATGAAACTGTATAATATTAAC (<400>18)), respectively, andsewing the products together via PCR using the primers PbF and PfR. Theinsertion of KpnI (underlined) and XbaI sites (boldface) into theoligonucleotides facilitated cloning of the resulting fragment into theKpnI/XbaI site of pGem4Z (Promega) and the hsp86 3′ untranslated region(UTR) from pHC2 (Crabb, B. S. (1997) supra). was cloned immediatelydownstream of this. The entire MSP-1/hsp86 3′ sequence, which was toserve as 5′ targeting sequence, was subsequently excised withKpnI/HindIII, the HindIII site filled in with Klenow reagent and thefragment cloned into the KpnI/lHindII site of pD_(B)D_(TmΔH)D_(B) (vanSpaendonk, R. M. L., Ramesar, J., van Wigcheren, A., Eling, W., Beetsma,A. L., van Gemert, G-J., Hooghof, J., Janse, C. J. and Waters, A. P.(2001) J. Biol Chem 276:22638-22647). A 0.55 Kb 3′ targeting sequence,comprising the P. berghei MSP-1 3′ UTR, was cloned into the EcoRV/BamHIsite of this vector to create pPb-PfM19. The MSP-1 3′ UTR was isolatedby screening a P. berghei ANKA gDNA library (Pace, T., Birago, C.,Janse, C. J., Picci, L. and Ponzi, M. 1998. Mol Biochem Parasitol97:45-53) using the P. berghei MSP-1₁₉ sequence as a probe. This enabledthe design of oligonucleotides PbM3′F(5′-GGCGATATCATAAATTATTGAAATATTTGTTGGA (<400>19)) and PbM3′R(5′-CGCGGATCCTATACAAAACATATACAAC (<400>20)), which were used to PCRamplify the P. berghei MSP-1 3′ UTR from P. berghei gDNA. The plasmidpPb-PbM19 is analogous to that of pPb-PfM19 with the exception that theentire MSP-1 5′ targeting sequence is that of P. berghei. This fragmentwas amplified from P. berghei ANKA gDNA using the oligonucleotides PbFand PbR2 (5′-TGCTCTAGATTAAAATATATTAAATACAAT-TAATGTG (<400>21)).

P. berghei Transfection

Eight week old Balb/c mice were used for the infection of P. bergheiANKA parasites. Infected blood at a 5% parasitemia was cultured in vitrousing standard procedures (Janse, C. J., Mons, B., Croon, J. J. A. B.and van der Kaay, H. J. (1984) Int J Parasitol 14:317-320) and purifiedschizonts were used for the electroporation of pPb-PbM19 and pPb-PfM19that had been linearised at the ends of the 5′ and 3′ targetingsequences using the restriction enzymes BamHI and ClaI (de Koning-Ward,T. F., Janse, C. J. and Waters, A. P. (2000). Annu Rev Microbiol54:157-185). The resulting transfection mix was inoculated intravenously(i.v) into 2 Balb/c mice and transgenic parasites were selected usingpyrimethamine (10 mg/kg bodyweight) as previously described (Menard, R.,and Janse, C. J. (1997). Enzymol 13:148-159).

Nucleic Acid Analysis

Genomic DNA (gDNA) was extracted from asynchronous parasite-infectedmouse blood after leukocyte removal on a CF-11 cellulose column(Whatman). PCR amplification and analysis of nucleic acids by Southernblotting was performed using standard methodologies (Sambrook, J. et al.(1989) supra).

MSP-1₁₉ GST Fusion Proteins and Generation of Antisera

The DNA sequence corresponding to the MSP-1₁₉ fragment lacking the GPIanchor sequence (amino acid Gly 1672-Ser 1766) was amplified from P.berghei ANKA gDNA using the oligonucleotides PbM19eF(5′-CGCGGATCCGGTATAGACCCTAAGCATGTATG (<400>22)) and PbM19eR(5′-GGAAGATCTTAGCTACAGAATACACCATCATAAT (<400>23)). The resulting PCRproduct was ligated into the BamHI site of pGEX-4T-1 and expressed as aglutathione S-transferase (GST) fusion protein (termed GST-PbM19) andrabbit antisera to GST-PbM19 was derived as described previously(O'Donnell, R. A., de Koning-Ward, T. F., Burt, R. A., Bockarie, M.,Reeder, J. C., Cowman, A. F. and Crabb, B. S. (2001). J Exp Med193:1403-1412).

Western Blot and Indirect Immunofluorescence Assay (IFA)

P. berghei-infected mouse blood was cultured in vitro to obtain culturesenriched for schizonts and merozoites. Parasites were analysed bywestern blot and IFA using rabbit polyconal antibodies raised againstGST-PbM19 and GST-PfM19 fusion proteins and a P. falciparumMSP-1₁₉-specific monoclonal antibody 4H9/19 as described (O'Donnell, R.A. et al. (2001) supra; Cooper, J. A. et al. (1992) supra; O'Donnell, R.A., Saul, A., Cowman, A. F. and Crabb, B. S. (2000) Nat Med 6:91-95.).

Generation of Semi-immune Mice

Semi-immune Balb/c mice were generated by the administration of 1×10⁴erythrocytes infected with either the Pb-PbM19 or Pb-PfM19 chimericline. When the parasitemia of these mice reached approximately 5-10%they were treated for 5 consecutive days with chloroquine (CQ) (10 mg/kgbodyweight). Recrudescence was typically observed 1 week after thisprimary infection after which mice were administered another 5 doses ofCQ. One month later mice were experimentally re-infected and then drugcured as above. Sera were obtained from individual mice 10 days afterthe final drug treatment to monitor MSP-1₁₉ antibodies. On the day ofchallenge (3 days after being bled for serology) blood smears wereexamined for parasites to ensure that mice were not infected withrecrudescing parasites. For challenge infections, mice were injected i.pwith 5×10⁶ Pb-PbM19 or Pb-PfM19 infected erythrocytes and the course ofparasitemia was monitored by microscopic examination of Giemsa stainedblood smears.

Serology

Antibodies reacting with recombinant P. berghei or P. falciparum MSP-1₁₉were detected by ELISA as previously described (O'Donnell, R. A. et al.(2001) supra). Blood taken from mice prior to primary infection wereused as negative controls in the ELISA. The optical density (OD) wasread at 450 nm and the ELISA endpoint titres taken as the highest serumdilution that gave an OD reading 5 times above that of the control sera.Inhibition of invasion assays using the P. falciparum lines D10-PfM3′and D10-PcMEGF were performed as described previously (O'Donnell, R. A.et al. (2001) supra).

Results

Allelic Replacement of P. berghei MSP-1₁₉ with P. falciparum MSP-1₁₉:Functional Complementation of Divergent MSP-1₁₉ Sequences

To establish whether P. falciparum MSP-1₁₉ can complement the in vivofunction of the divergent P. berghei MSP-1₁₉ domain, we sought to createa P. berghei MSP-1 chimera that expresses P. falciparum MSP-1₁₉ in placeof the endogenous molecule (FIG. 7). For this purpose, the transfectionvector pPb-PfM19 was constructed. This plasmid was designed to integrateinto the P. berghei MSP-1 locus by double-crossover homologousrecombination in a manner that results in replacement of endogenoussequences encoding epidermal growth factor (EGF) domains 1 and 2, inaddition to the GPI recognition sequence, with the corresponding P.falciparum (D10 line) sequence (FIGS. 7 and 8A). A second plasmid,pPb-PbM19, designed to integrate in an identical manner but resulting ina homologous MSP-1₁₉ replacement was also constructed to generate acontrol transfectant. Both plasmids were electroporated into the P.berghei (ANKA) line and parasites surviving 2 passages in mice underpyrimethamine selection were cloned by limiting dilution and analysedfurther. Southern blot analysis of gDNA showed that integration hadoccurred in these parasites by the expected double crossover event intoMSP-1 (FIG. 8). The resulting P. berghei/P. falciparum chimeric line,which we have termed Pb-PfM19, could be distinguished from a control P.berghei transfection line, termed Pb-PbM19, by restriction endonucleasedigestion with PstI (FIG. 8B). In addition, PCR amplification of gDNAusing oligonucleotides specific for an integration event into MSP-1 gavethe expected size products, which upon sequencing, confirmed that theexpected integration event had occurred (data not shown).

To determine whether the Pb-PfM19 and Pb-PbM19 lines expressed theexpected MSP-1₁₉ domains, western blot analysis was performed on latestage parasite extracts using domain specific anti-MSP-1₁₉ antibodies(FIG. 9A). The antibodies specific for P. falciparum MSP-1₁₉ recognisedboth MSP-1₁₉ and an ˜200 kDa band corresponding to full-length MSP-1 inPb-PfM19 parasites but not in Pb-PbM19 parasites. In contrast,antibodies specific for P. berghei MSP-1₁₉ only recognised MSP-1₁₉ andfull-length MSP-1 in wildtype P. berghei and the transfection controlline, Pb-PbM19. This demonstrates that P. falciparum MSP-1₁₉ can becorrectly expressed and processed in P. berghei and that the endogenousMSP-1₁₉ gene is no longer expressed in Pb-PfM19 parasites. Thelocalisation of MSP-1₁₉ in P. berghei lines was also assessed bydouble-labelling IFA. Characteristic merozoite surface labelling wasobserved in both chimera lines, with Pb-PfM19 parasites reacting onlywith the P. falciparum specific monoclonal antibody 4H9/19 while P.berghei wildtype and Pb-PbM19 chimeric parasites reacted only withrabbit anti-P. berghei MSP-1₁₉ antibodies (FIG. 9B). This confirms thatthe appropriate MSP-1₁₉ domain is correctly localised in both Pb-PbM19and Pb-PfM19 parasite lines. The growth rates of the transfected lineswere also examined and compared to the wildtype parasite line (FIG. 9C).All mice injected with 1×10⁴ parasites succumbed to infection over asimilar time frame, regardless of which parasite line they were given.These results extend our previous finding that the function of MSP-1₁₉during in vitro culture is conserved across divergent Plasmodium species(9, 21) to show that MSP-1₁₉ function is also conserved during theerythrocytic cycle in vivo.

A key rolefor MSP-1₁₉-specific Invasion Inhibitory Antibodies inProtection Elicited by Repeated Infection/drug Cure

Sera from BALB/c mice that were rendered semi-immune to either Pb-PfM19or Pb-PbM19 as a result of a low dose infection/drug cure regimen weretested for total MSP-1₁₉ antibodies in ELISA and for MSP-1₁₉ specificinvasion inhibitory antibodies using a novel P. falciparum in vitrogrowth assay (O'Donnell, R. A. et al. (2001) supra). All mice generateda strong MSP-1₁₉ antibody response that was specific for the relevantMSP-1₁₉ domain (FIG. 10A). This data highlights the immunogenicity ofthis domain in the context of a low dose blood-stage infection procedureand validates the expression of the appropriate MSP-1₁₉ domains in thetransfected P. berghei lines.

For the in vitro inhibition assay, the ability of a given serum toinhibit the invasion of RBC by two isogenic P. falciparum lines,D10-PfM3′ and D10-PcMEGF (FIG. 7), that differ only in their MSP-1₁₉domains was compared. D10-PcMEGF expresses the antigenically diverse P.chabaudi MSP-1₁₉ polypeptide and so is not recognised by P. falciparumMSP-1₁₉ specific antibodies. Hence, P. falciparum MSP-1₁₉ specificinvasion inhibitory activity of a given serum can be calculated bydetermining the difference in invasion rates of D10-PfM3′, whichutilises the wt P. falciparum MSP-1₁₉ domain for invasion, andD10-PcMEGF in the presence of the test serum. All sera from Pb-PfM19mice inhibited D10-PfM3′ parasites far more effectively than D10-PcMEGFparasites (FIG. 10B). Conversely, all sera from Pb-PbM19 immune miceinhibited D10-PcMEGF more effectively than wt P. falciparum. Since P.chabaudi and P. berghei are closely related rodent parasites withsomewhat conserved MSP-1₁₉ domains (73% identity) a degree of antigeniccross-reactivity was expected here. However, many epitopes differbetween the MSP-1₁₉ domains of rodent malaria parasites (Benjamin, P.A., Ling, I. T., Clottey, G., Spencer, Valero, L. M., Ogun, S. A.,Fleck, S. L., Walliker, D., Morgan, W. D., Birdsall, B., Feeney, J. andHolder, A. A. (1999). Mol Biochem Parasitol 104:147-156), hence theinvasion-inhibitory activity of these sera cannot be accuratelydetermined.

To determine if there is an association between the levels of MSP-1₁₉specific invasion inhibitory antibodies present in mouse serum anddegree of protection from a subsequent parasite challenge, Pb-PfM19semi-immune mice were administered a high dose (5×10⁶) of Pb-PfM19infected erythrocytes 3 days after they had been bled for theserological analyses described above. Following challenge, the course ofparasitemia was determined and plotted against levels of P. falciparumMSP-1₁₉-specific invasion inhibitory antibodies (FIG. 11). Strongevidence of regression was observed (R^(2=0.63); P=0.01 by Anova),implicating a substantial role for MSP-1₁₉ specific inhibitoryantibodies in controlling a blood-stage infection. A similarlysignificant regression curve was evident when invasion inhibition wasplotted against cumulative parasitemia in these mice (R²=0.56; P=0.02).Also, a 2-sided Rank Correlation Test, a more stringent analysis thatdoes not assume a linear relationship between 2 parameters, alsodemonstrated significance for peak parasitemia versus MSP-1₁₉-specificinvasion inhibition (P=0.05). Importantly, all mice had very similaranti-PfMSP-1₁₉ ELISA antibody endpoint titres (FIG. 10A), hence, noassociation between IgG levels and protection were observed.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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van Spaendonk, R. M. L., Ramesar, J., van Wigcheren, A., Eling, W.,Beetsma, A. L., van Gemert, G.-J., Hooghof, J., Janse, C. J. and Waters,A. P. (2001) J Biol Chem. 276:22638 TABLE I Reactivity in ELISA of Serafrom Malaria-infected Humans and Mice Against Recombinant P. falciparumand P. chabaudi MSP-1₁₉ Mean OD_(450 nm)* Sera n GST-PfM19 GST-PcM19PNG-B 47 0.846 ± 0.200 0.072 ± 0.158 PNG-M 33 0.481 ± 0.374 0.022 ±0.032 Pc immune 6 0.016 ± 0.012 0.613 ± 0.197*Mean OD readings after subtraction of GST reactivity ± SD. All serawere tested at a 1:3,200 dilution.‡ Number of sera above an OD value which equalled the mean plus threeSDs of that registered in replicate assays with pooled normal human ormouse sera where relevant.The cut-off values ranged from 0.078 to 0.180 depending on theserum/antigen combination.

1. A method of detecting the presence of a functionally modulatoryimmunointeractive molecule in a biological sample, whichimmunointeractive molecule is directed to a pathogen derived antigen,said method comprising: (i) contacting a pathogen expressing anepitopically distinct form of said antigen with said sample for a timeand under conditions sufficient to facilitate immunointeraction; (ii)contacting a pathogen expressing an epitopically native form of saidantigen with said sample for a time and under conditions sufficient tofacilitate immunointeraction; (iii) assessing the level of functionalactivity of the pathogens of step (i) and step (ii) wherein modulationin the functional activity of the pathogen of step (ii) relative to thepathogen of step (i) is indicative of the presence of a functionallyinhibitory immunointeractive molecule in said sample.
 2. The methodaccording to claim 1 wherein said modulation is down-regulation.
 3. Themethod according to claim 1 or 2 wherein said immunointeractive moleculeis an antibody.
 4. The method according to claim 1 or 2 or 3 whereinsaid method is performed in vitro.
 5. The method according to claim 4wherein said pathogen is a parasite.
 6. The method according to claim 1or 2 or 3 wherein said method is performed in vivo.
 7. The methodaccording to claim 6 wherein said pathogen is a parisite.
 8. The methodaccording to claim 5 or 7 wherein said parasite is a Plasmodium species.9. The method according to claim 8 wherein the Plasmodium species towhich said antibody is directed is one of Plasmodium falciparum,Plasmodium malariae, Plasmodium ovare or Plasmodium vivax.
 10. Themethod according to claim 9 wherein said antigen is any domain of MSP-1.11. The method according to claim 10 wherein said domain of MSP-1 is theblock 2 N-terminal domain or the block 17 C-terminal domain.
 12. Themethod according to claim 9 wherein said antigen is the apical membranedomain (AMA-1).
 13. The method according to claim 9 wherein said antigenis the merozoite surface protein 2, 3, 4 or 5 (MSP-2, MSP-3, MSP-4 orMSP-5).
 14. The method according to claim 9 wherein said antigen is therhoptry associated protein 2 (RAP-2).
 15. The method according to claim9 wherein said antigen is the erythrocyte binding antigen (EBA-175). 16.The method according to claim 9 wherein said antigen is thecircumsprozoite antigen (CSP).
 17. The method according to claim 4 or 6wherein said immunointeractive molecule is directed to a Plasmodiumchabaudi antigen, said pathogen of step (i) is wild-type Plasmodiumfalciparum and said pathogen of step (ii) is Plasmodium falciparumexpressing said Plasmodium chabaudi antigen.
 18. The method according toclaim 17 wherein said antigen is MSP-1₁₉, said Plasmodium falciparumpathogen of step (i) is the strain D10 and said Plasmodium falciparumpathogen of step (ii) is the strain D10-P MEGF or D10-PcM3′.
 19. Themethod according to claim 6 wherein said method is performed in vivo inmice, said immunointeractive molecule is directed to a Plasmodiumfalciparum antigen, said pathogen of step (ii) is wild-type Plasmodiumberghei and said pathogen of step (ii) is Plasmodium berghei expressingsaid Plasmodium falciparum antigen.
 20. The method according to claim 19wherein said antigen is MSP-1₁₉ and said Plasmodium berghei of step (ii)is the strain Pb-PfM19.
 21. An isolated malaria pathogen expressing anon-wild-type form of one or more antigens derived from said pathogen.22. The malaria pathogen of claim 21 wherein said pathogen is P.falciparum.
 23. The Plasmodium falciparum of claim 22 wherein saidantigen is MSP-1₁₉.
 24. The Plasmodium falciparum of claim 23 whereinsaid MSP-1₁₉ antigen corresponds to all or part of the Plasmodiumchabaudi MSP-1₁₉ region.
 25. The Plasmodium falciparum according toclaim 24 corresponding to strain D10-PcMEGF.
 26. The Plasmodiumfalciparum according to claim 24 corresponding to strain D10-PcM3′. 27.The malaria pathogen of claim 21 wherein said pathogen is Plasmodiumberghei.
 28. The Plasmodium berghei of claim 27 wherein said antigen isMSP-1₁₉.
 29. The Plasmodium berghei of claim 28 wherein said antigencorresponds to all or part of the Plasmodium falciparum MSP-1₁₉ region.30. The Plasmodium berghei according to claim 29 corresponding to strainPb-PfM19.
 31. A method of assessing any one or more qualitative and/orquantitative aspects of an immune response directed to a pathogen inaccordance with the method of any one of claims 1-20.